A Pilot Study of ¹⁸F-Alfatide PET/CT Imaging for Detecting Lymph Node Metastases in Patients with Non-Small Cell Lung Cancer

Abstract

Angiogenesis plays a key role in tumor development and α_vβ₃ integrin are overexpressed on the endothelial cell surface of newly forming vessels. ¹⁸F-Alfatide has favorable properties for α_vβ₃ integrin targeting and showed potential for imaging angiogenesis with Positron Emission Tomography (PET)/computed tomography (CT). In this study, 13 patients with non-small cell lung cancer (NSCLC) who underwent ¹⁸F-Alfatide PET/CT before surgery were enrolled. The uptake of all dissected lymph nodes (LNs) of ¹⁸F-Alfatide were assessed visually and analyzed with a maximum and mean standard uptake value (SUV_max, SUV_mean) and SUV ratios. LN metastases were pathologically confirmed and 20 of 196 LNs were malignant. All malignant LNs were successfully visualized on ¹⁸F-Alfatide PET/CT in patients and the sensitivity, specificity and accuracy was 100.0%, 94.9% and 95.4%, respectively. SUV_max, SUV_mean and SUV ratios in malignant LNs were significantly higher than in benign LNs for NSCLC patients (P < 0.001). The same result was observed in patients with adenocarcinoma and squamous cell carcinoma (P < 0.001). The ¹⁸F-Alfatide parameter shows high sensitivity (83.9–100%), specificity (78.6–96.7%) and accuracy (81.7–96.9%) according to thresholds calculated from receiver operating characteristic curve. Our results suggest that ¹⁸F-Alfatide PET/CT is valuable in the diagnosis of metastatic LNs for NSCLC patients.

Introduction

Preoperative staging of mediastinal lymph nodes (MLNs) provides accurate information on the extent of non-small cell lung cancer (NSCLC), it determines the prognosis and guides the choice of therapeutic modalities, consequently is of great importance for patients with NSCLC¹. As a result, the demand for noninvasive imaging is increased to promote MLN-staging accuracy in NSCLC patients.

¹⁸F-AlF-NOTA-PRGD₂ (¹⁸F-Alfatide), a novel tracer targeting integrin α_vβ₃, has been studied for angiogenesis imaging by positron emission tomography (PET)^{2, 3}. The growth of neovascularization from preexisting ones is called angiogenesis and angiogenesis is a major way in tumor growth and metastases⁴. In the integrin family, one of the most critical molecules involving in tumor angiogenesis and metastases is integrin receptor α_vβ₃ ^{5, 6}. The integrin α_vβ₃ which from a class of transmembrane glycoproteins consisting of 18α- and 8β- subunits is researched the most widely and is significantly up-regulated in activated endothelial cells of tumors undergoing angiogenesis, but not expressed in normal cells and quiescent vessel cells^{3, 4, 7}. Therefore, tumor angiogenesis can be evaluated by imaging α_vβ₃ expression, making the integrin receptor α_vβ₃ a valuable target for diagnosing malignant tumors and metastases³.

The various modifications of cyclic arginine-glycine-aspartic acid (RGD) peptides have been labeled with ^99mTc⁸ and ¹¹¹In⁹ for SPECT imaging, and with ¹⁸F¹⁰, ⁶⁴Cu¹¹, ⁶⁸Ga^{12, 13} and ⁸⁹Zr¹⁴ for PET imaging because integrin α_vβ₃ can interact with several extracellular matrix (ECM) proteins through the RGD tri-peptide sequence. ¹⁸F-Alfatide, a new one-step labeled integrin α_vβ₃-targeting PET probe, is simple and time-saving when synthesized compared with various ¹⁸F-labelled RGD peptide tracers³, including ¹⁸F-galacto-RGD^15–18, ¹⁸F-AH111585^{19, 20}, ¹⁸F-RGD-K5²¹, ¹⁸FFPRGD2 and ¹⁸F-FPPRGD2²².

In a previous clinical study, this new tracer, ¹⁸F-Alfatide, is safe when used in the human body and possibly valid in diagnosing primary tumors in patients with NSCLC³. Therefore, ¹⁸F-Alfatide was used as a novel tracer for integrin α_vβ₃ PET/CT examination in this present research. The objective was to detect lymph node metastases (LNMs) in patients with NSCLC, and to conduct a pilot research of ¹⁸F-Alfatide PET/CT diagnostic ability in the imaging of mediastinal LNMs in patients with NSCLC.

Results

After the examination of ¹⁸F-Alfatide PET/CT, no clinically detectable pharmacologic effects or adverse reactions were observed in any of these subjects. There were no marked changes in laboratory values or vital signs.

Twenty of the resected 196 lymph nodes (10.2%) were malignant in patients with NSCLC, six of 104 lymph nodes were malignant in patients with adenocarcinoma (AC) and 5 of the resected 65 lymph nodes were malignant in patients with squamous cell carcinoma (SCC). All the metastatic lymph nodes could clearly be delineated visually among the resected lymph nodes on the ¹⁸F-Alfatide PET/CT images. Receiver operating characteristic (ROC) curves for semi-quantitative assessment were illustrated by Fig. 1(a,b,c). Figure 2 the upper row provides an example of lymph node metastasis proved by gold standard and the lower row provides a false positive example.

Figure 1.

Receiver operating characteristic curves for the semi-quantitative assessment in patients with NSCLC (a), adenocarcinoma (b) and squamous cell cancer (c) at ¹⁸F-Alfatide PET/CT.

Figure 2.

The upper row shows a 62-year-old male suffering from an adenocarcinoma of the upper right lobe. LN station 10 according to Mountain and Dresler¹ was classified as a true positive case (a,b,c,d). The lower row shows a 59-year-old male suffering from a squamous cell carcinoma of the lower left lobe. The increased focal ¹⁸F-Alfatide uptake in PET/CT imaging is an example of false positive lymph node metastasis (e,f,g,h).

Lymph Nodes in NSCLC

Visual assessment

Using ¹⁸F-Alfatide, 20 lymph nodes were correctly recognized in accordance with pathological results and 9 were incorrectly found. Corresponding sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and accuracy were 100.0%, 94.9%, 100.0%, 69.0% and 95.4%.

Semi-quantitative assessment

Using ¹⁸F-Alfatide, malignant lymph nodes (median, 2.15; range, 1.1 to 3.8) had significantly higher maximum standardized uptake value (SUV_max) (P < 0.001) than benign lymph nodes (median, 0.9; range, 0.3 to 3.3) in patients with NSCLC. The same result was obtained for average SUV (SUV_mean) in distinguishing between malignant lymph nodes (median, 1.7; range, 0.9 to 2.5) and benign lymph nodes (median, 0.8; range, 0.3 to 2.6; P < 0.001). Table 1 shows statistically significant differences between benign and malignant lymph nodes for SUV_max, SUV_mean, and SUV ratios.

Table 1.

Characteristics of lymph node stations at ¹⁸F-Alfatide PET/CT.

Variables	NSCLC			AC			SCC
	Malignant	Benign	P	Malignant	Benign	P	Malignant	Benign	P
SUVmax	2.5(1.1–3.8)	0.9(0.3–3.3)	<0.01	1.8(1.1–3.8)	0.95(0.3–2.5)	<0.01	1.7(1.4–2.2)	0.9(0.4–3.3)	<0.01
SUVmean	1.7(0.9–2.5)	0.8(0.3–2.6)	<0.01	1.45(0.9–2.5)	0.9(0.3–1.8)	<0.01	1.3(1.1–1.8)	0.8(0.4–2.6)	<0.01
SUVLN/PT	0.5(0.2–0.9)	0.2(0.1–1.0)	<0.01	0.31(0.2–0.6)	0.24(0.1–1.0)	<0.01	0.3(0.27–0.4)	0.2(0.1–0.6)	<0.01
SUVLN/MBP	2.2(0.8–3.5)	0.9(0.3–3.0)	<0.01	1.1(0.8–3.2)	0.8(0.4–2.2)	<0.01	1.9(1.6–2.0)	1.0(0.4–3.0)	<0.01
SUVLN/M	4.1(2.2–9.5)	1.7(0.6–4.7)	<0.01	3.0(2.2–9.8)	1.7(0.6–4.2)	<0.01	3.0(2.8–3.4)	1.6(0.8–4.7)	<0.01

All data represent median (range). NSCLC, non-small cell lung cancer; AC, adenocarcinoma; SCC, squamous cell carcinoma; P, P value; SUV, standardized uptake value; LN, lymph node; MBP, mediastinal blood pool; PT, primary tumor; M, muscle.

The optimal cut-off values of SUV_max and SUV_mean were >1.4 and >1.2 by the ROC analyses (Fig. 1a; Table 2) and their respective areas under the curve (AUCs) were 0.95(95% confidence interval [CI], 0.91 to 0.98) and 0.94 (95% CI, 0.90 to 0.97), respectively. All results are represented in Table 3.

Table 2.

Receiver operating characteristic analysis: area under the curves and optimal cut-off values.

Parameter	NSCLC			AC			SCC
	AUC(95%CI)	Cut-off value	P	AUC(95%CI)	Cut-off Value	P	AUC(95%CI)	Cut-off Value	P
SUVmax	0.95(0.91–0.98)	>1.4	0.002	0.86(0.78–0.92)	>1.4	0.02	0.98(0.91–1.0)	>1.3	0.14
SUVmean	0.94(0.90–0.97)	>1.2	0.008	0.83(0.75–0.90)	>1.1	0.05	0.97(0.89–1.0)	>1	0.29
SUVLN/PT	0.89(0.83–0.93)	>0.3		0.71(0.61–0.79)	>0.3		0.95(0.86–0.99)	>0.3
SUVLN/MBP	0.92(0.87–0.96)	>1.5	0.24	0.82(0.73–0.89)	>0.9	0.40	0.98(0.91–1.0)	>1.5	0.14
SUVLN/M	0.97(0.94–0.99)	>2.4	0.01	0.94(0.88–0.98)	>2.4	0.06	0.98(0.91–1.0)	>2.6	0.14

CI, confidence interval; SUV, standardized uptake value; LN, lymph node; MBPS, mediastinal blood pool; PT, primary tumor; M, muscle; P, P value.

Table 3.

Diagnostic performances of the SUV parameters at ¹⁸F-Alfatide PET/CT in patients with NSCLC.

NSCLC	Sensitivity		Specificity		PPV		NPV		Accuracy
	%	95%CI	%	95%CI	%	95%CI	%	95%CI	%	95%CI
Visual	100.0	83.2–100	94.9	90.5–97.6	69.0	49.2–84.7	100	97.8–100	95.4	91.5–97.9
SUVmax	90.0	68.3–98.2	96.0	92.0–98.4	72.0	50.6–87.93	98.8	95.4–99.9	95.4	91.5–97.9
SUVmean	85.0	62.1–96.8	92.1	87.0–95.6	54.8	36.0–72.7	98.2	94.8–99.62	91.3	86.5–94.9
SUVLN/PT	85.0	62.1–96.8	81.2	75.3–87.2	34.7	21.7–49.6	98.0	94.2–99.6	82.1	76.1–87.2
SUVLN/MBP	85.0	62.1–96.8	96.0	92.0–98.4	70.8	48.9–87.4	98.3	95.0–99.6	94.9	90.8–97.5
SUVLN/M	100.0	83.2–100	89.2	83.7–93.4	51.3	34.8–67.6	100	97.7–100	90.3	85.3–94.1

NSCLC, non-small cell lung cancer; LN, lymph node; MBPS, mediastinal blood pool; M, muscle; PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval; SUV, standardized uptake value.

Subgroups of NSCLC

Visual assessment of lymph nodes in AC

11 lymph nodes (10.6%) in patients with AC were recognized visually and six of them (54.5%) were confirmed as pathologically malignant, while five of the 11 lymph nodes (45.6%) were proven to be false positive. Corresponding sensitivity, specificity, NPV, PPV and accuracy were 100.0%, 94.9%, 100.0%, 54.6% and 95.2% (Table 4).

Table 4.

Diagnostic performances of the SUV parameters at ¹⁸F-Alfatide PET/CT in patients with AC.

AC	Sensitivity		Specificity		PPV		NPV		Accuracy
	%	95%CI	%	95%CI	%	95%CI	%	95%CI	%	95%CI
Visual	100.0	54.1–100	94.9	88.5–98.3	54.6	23.4–83.3	100	96.1–100	95.2	89.1–98.4
SUVmax	83.3	35.9–99.6	94.9	88.5–98.3	50.0	18.7–81.3	98.9	94.2–100	94.2	87.9–97.9
SUVmean	100.0	54.1–100	80.6	71.4–87.9	24.0	9.4–45.1	100.0	95.4–100	81.7	73.0–88.6
SUVLN/PT	66.7	22.3–95.7	78.6	69.1–86.2	16.0	4.5–36.1	97.5	95.2–99.7	77.9	68.7–85.4
SUVLN/MBP	83.3	35.9–99.6	68.4	58.2–77.4	13.9	4.7–29.5	98.5	92.1–100	69.2	59.4–77.9
SUVLN/M	100	54.1–100	91.8	84.6–96.4	42.9	17.7–71.1	100	95.4–100	92.3	85.4–96.6

AC, adenocarcinoma; PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval; SUV, standardized uptake value; LN, lymph node; MBPS, mediastinal blood pool; M, muscle.

Semi-quantitative assessment of lymph nodes in AC

Using ¹⁸F-Alfatide, the malignant lymph nodes (median, 1.8; range, 1.1 to 3.8) has significantly higher SUV_max (P < 0.001) than in benign lymph nodes (median, 0.95; range, 0.3 to 2.5). The same result was obtained for the SUV_mean in distinguishing between malignant lymph nodes (median, 1.45; range, 0.9 to 2.5) and benign lymph nodes (median, 0.9; range, 0.3 to 1.8; P < 0.001). Table 1 shows statistically significant differences between benign and malignant lymph nodes for SUV_max, SUV_mean, and SUV ratios.

The optimal cut-off values of SUV_max and SUV_mean were >1.4 and >1.1 by the ROC analyses (Fig. 1b; Table 2) and their AUCs were 0.86 (95% CI, 0.78 to 0.92) and 0.83 (95% CI, 0.75 to 0.90), respectively. All results are represented in Table 4.

Visual assessment of lymph nodes in SCC

9 lymph nodes (13.8%) in patients with SCC were recognized visually and five of them (55.6%) were confirmed as pathologically malignant, while four of the 9 lymph nodes (44.4%) were proven as false positive. Corresponding sensitivity, specificity, PPV, NPV and accuracy were 100.0%, 93.3%, 55.6%, 100.0% and 93.9% (Table 5).

Table 5.

Diagnostic performances of the SUV parameters at ¹⁸F-Alfatide PET/CT in patients with SCC.

SCC	Sensitivity		Specificity		PPV		NPV		Accuracy
	%	95%CI	%	95%CI	%	95%CI	%	95%CI	%	95%CI
Visual	100.0	47.8–100	93.3	83.8–98.2	55.6	21.2–86.3	100.0	93.6–100	93.9	85.0–98.3
SUVmax	100.0	47.8–100	96.7	88.5–99.6	71.4	29.0–96.3	100.0	93.8–100	96.9	89.3–99.6
SUVmean	100.0	47.8–100	85.0	73.4–92.9	35.7	12.8–64.9	100.0	93.0–100	86.2	75.3–93.5
SUVLN/PT	60.0	4.6–94.7	91.7	81.6–97.2	37.5	8.5–75.5	96.5	87.9–99.6	89.2	79.1–95.6
SUVLN/MBP	100.0	47.8–100	96.7	88.5–99.6	71.4	29.0–96.3	100.0	93.8–100	96.9	89.3–99.6
SUVLN/M	100.0	47.8–100	96.7	88.5–99.6	71.4	29.0–96.3	100.0	93.8–100	96.9	89.3–99.6

SCC, squamous cell carcinoma; SUV, standardized uptake value; LN, lymph node; MBPS, mediastinal blood pool; M, muscle; PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval.

Semi-quantitative assessment of lymph nodes in SCC

The malignant lymph nodes (median, 1.7; range, 1.4 to 2.2) had significantly higher SUV_max(P < 0.001) than benign lymph nodes (median, 0.9; range, 0.4 to 3.3). The same result was obtained for SUV_mean in distinguishing between malignant lymph nodes (median, 1.3; range, 1.1 to 1.8) and benign lymph nodes (median, 0.8; range, 0.4 to 2.6; P < 0.001).

The optimal cut-off values of SUV_max and SUV_mean were >1.3 and >1.0 by the ROC analyses (Fig. 1c; Table 2) and their AUCs were 0.98 (95% CI, 0.91 to 1.0) and 0.98 (95% CI, 0.90 to 1.0), respectively. All results are represented in Table 5.

Discussion

This is a pilot clinical research to evaluate the prognostic value of the ¹⁸F-Alfatide PET/CT in detecting mediastinal LNMs in patients with NSCLC. This new tracer, ¹⁸F-Alfatide, has been again proven safe when used in clinical trials, is convenient in the synthesis process, and has shown a significant diagnostic value of mediastinal LNMs.

FDG PET/CT is widely used for detecting LNMs in patients with NSCLC. In a previous meta-analysis, mean sensitivity and specificity of FDG PET/CT for LNMs detection were 69% and 95%²³. The FDG PET/CT had a relatively high specificity with low sensitivity²⁴ and the results were barely satisfactory. Therefore, the researchers focused their attention on the development of new diagnostic methods and new tracers of PET for detecting LNMs in patients with NSCLC. ¹¹C-choline²⁵, ¹⁸F-fluorothymidine(¹⁸F-FLT)²⁶, 4′-[methyl-¹¹C]-thiothymidine(4DST)²⁷, ¹⁸F-Alfatide are all PET tracers used to visualize various malignancies and LNMs.

In the previous study, the accuracy of ¹¹C-choline PET/CT of LNMs detection was 83.76% and the sensitivity was 100%. These results seem to be encouraging, but specificity (72%) was not good enough for diagnosing LNMs in patients with NSCLC^{25, 28}. Yamamoto et al.²⁶ indicated that the diagnostic ability of FLT PET was lower than FDG PET, the sensitivity, specificity, PPV, NPV and accuracy of FLT PET and FDG PET for lymph node staging were 57%, 93%, 67%, 89%, 85 and 57%, 78%, 36%, 91%, 74%, respectively. For 4DST, the results of clinical study suggested that the sensitivity for detecting LNMs was high (82%), but its low specificity (72%) was a limitation (P < 0.001)²⁷. Compared with FGD PET/CT and other tracers PET/CT, ¹⁸F-Alfatide PET/CT showed significantly high sensitivity (85–100%) and specificity (81.2–96.0%) for detecting mediastinal LNMs in patients with NSCLC. The sensitivity, specificity, PPV, NPV and accuracy of ¹⁸F-Alfatide PET/CT visual analysis were 100%, 94.9%, 69%, 100% and 95.4%, respectively.

Invasive surgical examinations, such as endobronchial ultrasonography transbronchial needle aspiration (EBUS-TBNA)²⁹ and mediastinoscopy, showed high specificity and sensitivity for lymph node staging. However, these tests are invasive, and lesion location may restrict the possibility of obtaining tissue samples. ¹⁸F-Alfatide PET/CT imaging is noninvasive and showed significantly high sensitivity and specificity for detecting mediastinal LNMs in patients with NSCLC. Among all ¹⁸F-Alfatide PET semi-quantitative parameters, the SUV_max showed a better performance and has the potential to serve as the most important semi-quantitative parameter to improve noninvasive nodal staging and treatment planning for LNMs after use of ¹⁸F-Alfatide PET/CT scans.

Angiogenesis is a vital process in tumor progression and tumor growth, and it is responsible for the metastasis of lymph nodes. Previous work suggests that high expression of integrin α_vβ₃ on the endothelial cells surface of angiogenesis is related to its proliferative and metastatic properties^30–32. Therefore, ¹⁸F-Alfatide is very sensitive to the growth and metastasis of tumors. The high sensitivity of visual and SUV_max (Tables 3, 4 and 5) for LNMs indicates that it may be detecting micro node metastases.

In this present study, seven AC cases (54%) and 4 SCC cases (31%) were included, and they showed a similar visual and semi-quantitative analysis outcome as the results from NSCLC patients. Even though the SUV_LN/PT was low in both AC and SCC, they did not show significant differences when compared with SUV_mean, SUV_LN/MBP and SUV_LN/M(P > 0.05). ¹⁸F-Alfatide PET/CT imaging showed a high sensitivity of SUV_max in patients with NSCLC (90.0%) and SCC (100%) but relatively low sensitivity (83.3%) in patients with AC. It is reported that AC is known for low FDG-avidity³³ and AC may have low affinity with ¹⁸F-Alfatide as well. This may explain the low sensitivity in AC.

¹⁸F-Alfatide PET/CT imaging appears to merit LNMs assessment, which is very important for clinical decision-making and surgical planning for NSCLC patients. Even though it has excellent results for lung cancer staging, 9 false-positives occurred in the present study. False-positive uptake was caused by chronic inflammatory and the inflammatory process often accompanying angiogenesis, which produces a large amount of integrin α_vβ₃ ³. The accuracy (95.4%; 95.2%; 93.9%) of visual analysis was similar with SUV_max (95.4%; 94.2%; 96.9%) in patients with NSCLC, AC and SCC. It is also of note that the simpler visual analysis is preferred and SUV would be a secondary aid.

The results indicated that ¹⁸F-Alfatide PET/CT imaging is potentially successful in diagnosing metastatic lymph nodes with a high sensitivity and specificity in patients with NSCLC. Even though the outcome of this present study is promising, the number of patients was small. Supplementary researches with a larger number of patients would be requested to confirm the results.

Methods

Thirteen patients including 196 assessable lymph nodes were analyzed (male, n = 6; female, n = 7; median age, 57 years [range, 45 to 69 years]). The including criteria: (1) histologically proven NSCLC, (2) all patients performed lobectomy + lymph node dissection surgery after ¹⁸F-Alfatide PET/CT imaging, (3) histological results of lymph nodes were available as the gold standard for diagnosis, (4) the age >18 years. Patients histological subtypes included SCC (n = 4), AC (n = 7) and NSCLC not otherwise specified (n = 2). Patient characteristics are represented in Table 6.

Table 6.

Patients information and diagnosis (n = 13).

Variable	NSCLC (n, %)
Total	13(100)
Age(years, range)	57(45–69)
Gender
male	6(46)
female	7(54)
Histology
adenocarcinoma	7(54)
Squamous Cell Carcinoma	4(31)
NSCLC,NOS	2(15)
N Stage (TNM)
0	8(62)
1	2(15)
2	3(23)
3	0(0)

NSCLC, non-small cell lung cancer; AC, adenocarcinoma; SCC, squamous cell carcinoma; NOS, not otherwise specified.

Approvals

This study was approved by the ethics committee of Shandong Cancer Hospital, and all patients provided written informed consent which included information on radiation exposure. All methods were carried out in accordance with relevant guidelines and regulations.

¹⁸F-Alfatide PET/CT imaging

All patients underwent an ¹⁸F-Alfatide PET/CT scan using an integrated PET/CT system (Discovery LS; GE Healthcare) from June 2013 to December 2016, which consisted a full-ring dedicated PET of and a spiral CT scan of the same axial range. We purchased PRGD2 peptide that could label with lyophilized kits simply from the Jiangsu Institute of Nuclear Medicine, and the synthesis process was carried out as previous studies^{2, 3}. The patients would perform ¹⁸F-Alfatide PET/CT scan without fasting or receiving CT contrast agents. The radiochemical purity of the ¹⁸F-Alfatide was higher than 95% after extraction, and its specific radioactivity was higher than 37 GBq (1000 mCi)/μmol. All patients would rest for approximately 60 minutes after injecting ¹⁸F-Alfatide (212.15 ± 30.8 MBq) intravenously. Axial views were reconstructed into sagittal and coronal views from the top of the neck to the upper abdomen. The patients had normal, shallow respirations during image acquisition. The images were attenuation corrected with the transmission data from CT. The Xeleris workstation (GE Healthcare) viewed the attenuation-corrected PET images, CT images, and fused PET/CT images as coronal, sagittal, and transaxial slices.

Image analysis

PET data was reconstructed using the ordered-subsets expectation maximization algorithm. The SUV was calculated according to the following formula: (measured activity concentration [Bq/mL] × body weight [g])/injected activity (Bq). Standard visual image interpretation and semi-quantitative analysis were conducted independently by 2 experienced nuclear medicine physicians who were blinded to the clinical and structural imaging findings. All the regions of interest were contoured separately and the SUV_max & SUV_mean of primary tumor, LNs, aorta and muscle were calculated. The increased ¹⁸F-Alfatide uptake regions were defined as positive when it showed definite uptake and not related to normal physiologic uptake. And the increased ¹⁸F-Alfatide uptake area was defined as negative when it was related to the physiologic biodistribution of ¹⁸F-Alfatide. Care was taken to exclude adjacent blood vessels. SUV ratios of lymph nodes to primary tumor, mediastinal blood pool and muscle were calculated by SUV_LN/PT, SUV_LN/MBP, SUV_LN/M assessment:

$$\begin{array}{lll}\hfill {\mathrm{SUV}}_{\mathrm{LN}/\mathrm{PT}}& \hfill =\hfill & {\mathrm{SUV}}_{\max }\left(\mathrm{LN}\right){/\mathrm{SUV}}_{\max }\left(\mathrm{primary}\mathrm{tumor}\right)\hfill \\ \hfill {\mathrm{SUV}}_{\mathrm{LN}/\mathrm{MBP}}& \hfill =\hfill & {\mathrm{SUV}}_{\max }\left(\mathrm{LN}\right){/\mathrm{SUV}}_{\max }\left(\mathrm{MBP}\right)\hfill \\ \hfill {\mathrm{SUV}}_{\mathrm{LN}/\mathrm{M}}& \hfill =\hfill & {\mathrm{SUV}}_{\max }\left(\mathrm{LN}\right){/\mathrm{SUV}}_{\max }\left(\mathrm{muscle}\right)\hfill \end{array}$$

Pathological analysis

All of the patients underwent lobectomy + lymph node dissection surgery. All resected lymph nodes were marked by surgeons during the surgery according to Mountain and Dresler¹. The tumor specimens were performed according to standard protocols as previously³. All hematoxylin-eosin staining tissue sections were reviewed by two pathologists who were blinded to image outcomes and arrived at a single, final conclusion between them. The pathological results were used as gold standard.

Statistical analysis

Statistical analyses were performed using the MedCalc software (MedCalc^®, version 15.2.2, 64-bit, MedCalc Software bvba, Ostend, Belgium). All semi-quantitative data are expressed as the median (range). Differences were considered statistically significant when two-tailed P values were less than 0.05. The significant differences between malignant and benign lymph nodes were tested by the Mann-Whitney U test for SUV_max, SUV_mean, SUV_LN/PT, SUV_LN/MBP and SUV_LN/M. The diagnostic performance of SUV parameters in differentiating malignant from benign LNs was analyzed using the ROC curves and AUCs with their 95% CIs. The optimal cut-off values of these variables producing maximum sensitivity plus specificity were determined from ROC analyses. The nonparametric method proposed by DeLong et al.³⁴ was used to compare correlated ROC curves by MedCalc software.

Author Contributions

Y.Z. wrote the manuscript with all authors commenting. Y.Z., S.G., Y.H., J.S.Z., Y.J.D., B.J.Z., S.Q.Z. and H.L. performed the experiment. S.H.Y., Y.Z., S.G., Y.J.D. and Y.H. participated in data analyses. Z.B.L. gave a critical review and edited the manuscript. S.H.Y., J.M.Y. and Y.Z. designed the research.

Competing Interests

The authors declare that they have no competing interests.