cT shows high specificity

Imaging

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Original Research n Thoracic

Integrin Imaging with 99mTc3PRGD2 SPECT/CT Shows High Specificity in the Diagnosis of Lymph Node Metastasis from Non–Small Cell Lung Cancer1 Xiaona Jin, MD Naixin Liang, MD Mengzhao Wang, MD Yunxiao Meng, MD Bing Jia, PhD Ximin Shi, MD Shanqing Li, MD Jinmei Luo, MD Yaping Luo, MD Quancai Cui, MD Kun Zheng, MD Zhaofei Liu, PhD Jiyun Shi, PhD Fang Li, MD Fan Wang, PhD Zhaohui Zhu, MD

Purpose:

To evaluate an integrin imaging approach based on single photon emission computed tomography (SPECT)/computed tomography (CT) by using technetium 99m (99mTc)dimeric cyclic arginine-glycine–aspartic acid (RGD) peptides with three polyethylene glycol spacers (3PRGD2) as the tracer to target the integrin avb3 expression in lung cancer and lymph node metastasis.

Materials and Methods:

With ethics committee approval and written informed consent, 65 patients (41 male, 24 female; mean age, 60 years 6 11 [standard deviation]) with suspicious lung lesions were recruited with informed consent. The patients underwent both 99mTc-3PRGD2 SPECT/CT and fluorine 18 (18F) fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT within 1 week. Finally, 65 lung lesions in 53 patients were pathologically diagnosed as non–small cell lung cancer (NSCLC) and 14 lung lesions in 12 patients were benign. Per-region analysis of lymph nodes included 248 regions with metastasis and 56 negative regions. Twenty specimens from the removed lung lesions or lymph nodes were stained with integrin avb3, CD34, and Ki-67 to correlate with the image findings. Receiver operating characteristic curve, z statistics, McNemar test, and x2 analysis were used to compare the diagnostic performance of the two imaging methods.

1

From the Departments of Nuclear Medicine (X.J., X.S., Y.L., K.Z., F.L., Z.Z.), Thoracic Surgery (N.L., S.L.), Respiratory Medicine (M.W., J.L.), and Pathology (Y.M., Q.C.), Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Shuaifuyuan, Wangfujing Street, Dongcheng District, Beijing 100730, China; Medical Isotopes Research Center, Peking University, Beijing, China (B.J., Z.L., J.S., F.W.); and Interdisciplinary Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China (F.W.). Received May 22, 2015; revision requested July 28; final revision received March 17, 2016; accepted April 19; final version accepted May 24. Address correspondence to Z.Z. (e-mail: [email protected]). Supported in part by grant from Ministry of Science and Technology of the People’s Republic of China (2011YQ17006710, 2012ZX09102301-018), Outstanding Youth Fund (81125011), Ministry of Education of the People’s Republic of China (311037), “973” projects (2011CB707705, 2013CB733802), Special fund for scientific research in the public interests of health (201402001), National Natural Science Foundation of China (81071189. 81171369, 81171370, 81271614, 81321003), and Capital Special Project for Featured Clinical Application (Z111107058811096, Z121107001012119). The funding sources had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Results:

Conclusion:

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Tc-3PRGD2 SPECT/CT was found to be more specific than 18F-FDG PET/CT in the per-region diagnosis of lymph node metastasis (specificity, 94.6% vs 75.0%; P = .008) when the sensitivity of the two methods was comparable (88.3% vs 90.7%; P = .557). There was no significant difference between the two methods in the per-lesion diagnosis of lung tumor (z = 0.82, P = .410). The accumulation level of 99mTc-3PRGD2 was found in positive correlation with the integrin avb3 expression (r = 0.84, P = .001) and microvessel density (r = 0.63, P = .011) in the tumors. 99m

Tc-3PRGD2 SPECT/CT shows high specificity in the diagnosis of lymph node metastasis from NSCLC, which may benefit surgical decision making for the patients. RSNA, 2016

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X.J. and N.L. contributed equally to this work. RSNA, 2016

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radiology.rsna.org n Radiology: Volume 281: Number 3—December 2016

THORACIC IMAGING: Integrin Imaging in the Diagnosis of Lymph Node Metastasis

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n the basis of prior studies, we have proposed technetium 99m-dimeric cyclic arginineglycine–aspartic acid (RGD) peptides with three polyethylene glycol spacers (99mTc-3PRGD2) as a tracer for single photon emission computed tomography (SPECT) oncology by targeting the integrin avb3 in tumors (1,2). Integrin avb3 is a transmembrane heterodimeric receptor that mediates cell-cell and cell-extracellular matrix adhesion (3). With high expression on both neovascular endothelial cells and tumor cells but with no or very low expression on quiescent vessel cells and other normal cells, the integrin avb3 expression is believed to be involved in angiogenesis and metastasis, the two very important characteristics of malignancies (4,5). Therefore, integrin avb3 may be a promising target for the diagnosis and staging of malignant tumors. A prior study has preliminarily pro ved the feasibility of 99mTc3PRGD2 SPECT for integrin avb3 imaging of lung cancer in patients (2). This study was prospectively designed to evaluate an integrin imaging approach based on SPECT/computed tomographic (CT) findings, by using 99m Tc-3PRGD2 as the tracer to target the integrin avb3 expression in lung cancer and lymph node metastasis.

Advances in Knowledge nn Integrin avb3 imaging with technetium 99m-dimeric cyclic arginineglycine–aspartic acid (RGD) peptides with three polyethylene glycol spacers (99mTc-3PRGD2) SPECT/CT is more specific than glucose analog imaging with fluorine 18 fluorodeoxyglucose (FDG) PET/CT in the diagnosis of lymph node metastasis from non–small cell lung cancer (NSCLC) (specificity, 94.6% vs 75.0%; P = .008). nn The accumulation level of Tc3PRGD2 is correlated with the integrin avb3 expression (r = 0.84, P = .001) and microvessel density (r = 0.63, P = .011) in the tumors. 99m

Materials and Methods This study was approved by the Ethics Committee of Peking Union Medical College Hospital and registered online at the U.S. National Institutes of Health ClinicalTrials.gov (NCT01737112). Written informed consent was obtained from each patient before the study.

Patients This is a single-center prospective diagnostic efficiency study of 99mTc-3PRGD2 SPECT/CT in lung cancer, with fluorine 18 (18F) fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT serving as the reference method. The inclusion criteria were (a) age of 30 years or older, (b) highly suspicious lung lesions at thoracic CT, and (c) availability of histologic and follow-up results. The main exclusion criteria were (a) claustrophobia or other conditions unsuitable for the examinations (no patient was excluded after enrollment) and (b) patients with biopsy-proven small cell lung cancer, who were excluded because this study was designed for evaluation of non–small cell lung cancer (NSCLC) (two patients with small cell lung cancer were excluded). The consecutive patients underwent 99mTc3PRGD2 SPECT/CT and 18F-FDG PET/ CT within 1 week, without any treatment between the intervals. Finally, 65 lung lesions in 53 patients were pathologically diagnosed as NSCLC and 14 lung lesions in 12 patients were benign. A total of 248 regions of mediastinal lymph nodes (classified by using the American Joint Committee on Cancer and the Union for International Cancer Implications for Patient Care nn RGD integrin imaging has higher specificity than FDG metabolism imaging for noninvasive diagnosis of lymph node metastasis from NSCLC. nn

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Tc-3PRGD2 SPECT/CT is a specific method for the diagnosis of lymph node metastasis from NSCLC, which may benefit the staging and surgical decision making.

Radiology: Volume 281: Number 3—December 2016 n radiology.rsna.org

Jin et al

Control standard) were diagnosed with metastasis and 56 regions were negative according to surgery records and pathologic reports. From February 2011 to October 2012, 65 patients (41 male, 24 female) were recruited with written informed consent. The patients were 31–78 years of age (mean age, 60 years 6 11 [standard deviation]), and there was no significant difference in age between the men (age range, 32–78 years; mean age, 60 years 6 12) and women (age range, 31–71; mean age, 60 years 6 12; P = .329). All patients completed the study with pathologic diagnosis and had been followed up until death or for more than 2 years (Table 1).

Tc-3PRGD2 SPECT/CT Synthesis of the precursor, kit preparation, and subsequent 99mTc-labeling were performed as previously described (1,2). The radiochemical purity was proved to be greater than 95% before clinical use. Each patient was intravenously injected with 99mTc3PRGD2 in a dosage of approximately 11.1 MBq (0.3 mCi) per kilogram of body weight, ranging from 532.8 to 99m

Published online before print 10.1148/radiol.2016150813 Content codes: Radiology 2016; 281:958–966 Abbreviations: FDG = fluorodeoxyglucose NSCLC = non–small cell lung cancer RGD = arginine-glycine–aspartic acid 99m Tc-3PRGD2 = technetium 99m-dimeric cyclic RGD peptides with three polyethylene glycol spacers T/B = tumor-to-background Author contributions: Guarantors of integrity of entire study, X.J., N.L., Y.M., X.S., S.L., F.L., F.W., Z.Z.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; agrees to ensure any questions related to the work are appropriately resolved, all authors; literature research, X.J., N.L., M.W., Y.M., X.S., J.L., Y.L., K.Z., F.W., Z.Z.; clinical studies, X.J., N.L., M.W., Y.M., X.S., J.L., Q.C., K.Z., J.S., F.L., Z.Z.; experimental studies, X.J., N.L., Y.M., B.J., X.S., J.L., K.Z., F.W.; statistical analysis, X.J., N.L., X.S., J.L., F.L., F.W., Z.Z.; and manuscript editing, X.J., N.L., Y.M., X.S., J.L., Z.L., F.L., F.W., Z.Z. Conflicts of interest are listed at the end of this article.

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Table 1 Basic Information and Final Diagnosis of the Recruited Patients Demographic or Clinical Characteristics Age (y) Range Mean 6 standard deviation No. of patients No. of men No. of women Diagnosis Malignant Adenocarcinoma Squamous cell carcinoma Benign Granuloma Chronic inflammation Tuberculosis

No. of Patients* 40–77 60 6 11 65 41 (63.1) 24 (36.9) 65 53 (81.5) 36 (55.4) 17 (26.2) 12 (18.5) 6 (9.2) 5 (7.7) 1 (1.5)

* Unless otherwise indicated, data are the number of patients and data in parentheses are percentages.

876.9 MBq (mean, 679.3 MBq 6 94.0). Tc-3PRGD2 SPECT/CT scans were obtained by using a Precedence SPECT/ CT system (ADAC Laboratories, Philips Medical Systems company, Milpitas, Calif). After intravenous injection of 99m Tc-3PRGD2, anterior and posterior planar scans of the whole body and SPECT imaging of the chest were performed successively, centering at 1 hour within a time window of 6 20 minutes to complete the imaging procedure (range, 49–76 minutes; mean, 60.1 minutes 6 5.8). Low-dose spiral CT of the chest (120 kV, 50 mAs) was used for attenuation correction and anatomic localization. Any related side effect during the examination was inquired and recorded in detail. 99m

F-FDG PET/CT The patients fasted for at least 4 hours, and their blood glucose levels measured less than 6.4 mmol/L before intravenous injection of 18F-FDG in a dosage of approximately 5.55 MBq (0.15 mCi) per kilogram of body weight. 18F-FDG PET/CT was performed by using a Biograph 64 Truepoint TrueV system (Siemens Medical Solutions, Knoxville, Tenn) 60 minutes 6 10 after 18F-FDG 18

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injection. Low-dose CT (120 kV, 50 mAs) and PET examinations (five to six bed positions, 2 minutes per bed) were performed successively from the midthigh to the skull base.

Image Analysis The 99mTc-3PRGD2 SPECT/CT and 18FFDG PET/CT images were transferred to a MMWP workstation (Siemens) for comparison and analysis by three nuclear medicine physicians (Y.L., F.L., and Z.Z., with more than 3 years of expertise in nuclear oncology; they were different from the physicians who previously recruited the patients and interpreted the images) through consensus reading and blinded to the history, other examinations, and pathologic diagnosis of the patients. Per-lesion analysis was performed for the diagnosis of lung tumor, whereas because of the difficulty to correlate the lymph nodes lesion by lesion between the images and pathologic diagnosis, per-region analysis was adopted for the diagnosis of lymph node metastasis. They reached a consensus by discussion when a discrepancy existed. For semiquantitative analysis, tumor-to-background (T/B) ratios of both SPECT and PET images were measured by the same physicians using a standardized method as described below: A volume-of-interest method was used to obtain the maximum values (gamma counts for SPECT and standard uptake values for PET) of the lung tumors, as well as the prominent lymph node in each mediastinal region. The contralateral lung without any lesion was used as the control for lung lesions, whereas the blood pool of the aortic arch was set as the control for lymph nodes. The mean values of the control volumes of interest were measured as background. The T/B ratio was calculated by dividing the two values. Immunohistochemical Analysis Immunohistochemical staining of avb3 integrin, CD34, and Ki-67 was performed in 11 lung cancer specimens, four metastatic lymph nodes, four benign lymph nodes, and one granuloma by using formalin-fixed, paraffin-embedded tissue sections and

the Envision method with appropriate pretreatment (6). The representative specimens were selected by a pathologist (Y.M.) according to the quality and quantity of the embedded tissue. Microvessel density was counted on the CD34-stained slides in three fields at a magnification of 3200. The proliferation index was calculated as (number of Ki-67-labeled cells/total number of cells) 3 100% at a magnification of 3200 in five representative regions of the tumor. Two experienced pathologists blinded to the imaging results (Y.M. and Q.C.) read the integrin avb3stained slides in consensus and rated the stain on a four-point scale: 0 = negative; 1 = mild stain; 2 = moderate stain; 3 = intense stain.

Statistical Analysis The analysis was performed with the use of Prism 5.0 (GraphPad, San Diego, Calif) software, as well as SPSS 19.0 (SPSS, Chicago, Ill) for the McNemar test. Continuous variables were summarized as means 6 standard deviation; categorical variables were described in numbers and percentages. The correlation between quantitative parameters was evaluated by Pearson correlation coefficient for data with normal distribution or Spearman correlation coefficient for data with skewed distribution. For the data with normal distribution, paired t test (parametric test) was used to compare the mean T/B ratios of 99mTc-3PRGD2 and 18FFDG from the same patients; unpaired t test was used to compare the mean T/B ratios between the NSCLC in different stage. Receiver operating characteristic curve analysis and z statistics were used to compare the diagnostic performance of the two methods (7). McNemar test and x2 test was used to compare the diagnostic values of 99mTc3PRGD2 SPECT/CT and 18F-FDG PET/ CT. All tests were two tailed, with the significance level at .05. Results No side effect related to 99mTc-3PRGD2 injection was reported in the patients. The images of both 99mTc-3PRGD2



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Figure 1

Figure 1: Distribution of 18F-FDG (left column) and 99mTc-3PRGD2 (right column) in the same lesions in two patients with lung cancer. A, B, In a 73-year-old man with low differentiated adenocarcinoma in left lower lung, the anterior part shows higher 18F-FDG uptake (arrow), whereas the posterior part has more 99mTc-3PRGD2 accumulation (arrow). C, D, In a 40-year-old woman with low differentiated adenocarcinoma in right upper lung, the central part of the tumor shows higher 18F-FDG uptake (arrow) but lower 99mTc-3PRGD2 accumulation (arrow).

SPECT/CT and 18F-FDG PET/CT were all of good quality. The two methods were compared in the diagnosis of lung cancer and mediastinal lymph node metastasis. The 99mTc-3PRGD2 and 18FFDG accumulations were correlated with the immunohistochemical staining of integrin avb3, CD34, and Ki-67.

Diagnosis of Lung Cancer A total of 65 lung lesions were detected in the 53 NSCLC patients (36 adenocarcinomas and 17 squamous cell carcinomas), with sizes ranging from 1.1 to 9.9 cm (mean, 3.5 cm 6 2.4). None of these tumors was missed with either 99m Tc-3PRGD2 SPECT/CT or 18F-FDG PET/CT in visual analysis. The T/B ratios of 99mTc-3PRGD2 accumulation were 1.3 to 12.1 (mean, 5.8 6 2.6), significantly lower than those of 18FFDG uptake (range, 1.5–62.5; mean, 14.7 6 9.4; P , .001) in a lesion-bylesion analysis. A significant but weak correlation was observed between the

T/B ratios of the two tracers in the tumors (r = 0.40; P , .001). The different biology can be obviously observed by the different distribution of the two tracers in the same tumors in some patients (Fig 1). A total of 14 lung lesions from 12 patients were pathologically diagnosed as benign (six granulomas, five chronic inflammations, and one tuberculosis). The size of the lesions ranged from 1.3 to 7.5 cm (mean, 3.6 cm 6 1.9). The T/B ratios of 99mTc-3PRGD2 and 18 F-FDG were 1.9 to 5.2 (mean, 4.2 6 2.6) and 1.6 to 37.7 (mean, 8.6 6 9.2), respectively. In contrast to those findings in NSCLC, there was no significant difference (P = .104) or correlation (r = 0.13, P = .669) between the T/B ratios of the two tracers in the benign lung lesions. The NSCLC tumors had significantly higher T/B ratios than the benign lung lesions on both 99mTc-3PRGD2 SPECT/ CT and 18F-FDG PET/CT images (P =


.047 and P , .001, respectively). Receiver operating characteristic curve analysis and z statistics indicated no significant difference between the two methods in the diagnosis of lung cancer (z = 0.82, P = .410) (Table 2). Statistical analysis showed higher sensitivity of 18F-FDG PET/CT in the detection of lung cancer (P = .039), but no significant difference between the two methods in the other diagnostic values (Table 3). In the 36 patients with stage III or IV NSCLC, the 99mTc-3PRGD2 accumulation in the primary tumor was significantly higher than that in stage I or stage II (5.9 6 2.6 vs 2.6 6 4.2, P = .038), whereas the 18F-FDG uptake had no such significant difference between them (13.6 6 6.7 vs 17.3 6 13.8, P = .154). The 99mTc-3PRGD2 accumulation was significantly lower in the six NSCLC patients without any metastasis than in those with metastases (3.7 6 1.9 vs 5.9 6 2.5, P = .008), whereas the 18F-FDG 961


uptake had no such significant difference between them (12.3 6 9.0 vs 14.6 6 11.7, P = .566).

Diagnosis of Mediastinal Lymph Node Metastasis In the 248 regions recognized as having metastatic lymph nodes and sized from 0.3 to 3.5 cm (mean, 1.5 cm 6 0.7), 240 (96.8%) were detected as 18 F-FDG–avid lymph nodes (T/B: 3.2 6 1.9). 99mTc-3PRGD2 accumulation was found in the same 240 regions, with T/B ratios of 2.1 6 0.9, significantly lower than those of 18F-FDG uptake (P , .001). A significant correlation was observed between the uptake of the two tracers in the metastatic lymph nodes (r = 0.49; P , .001).

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According to the pathologic diagnosis, 56 hilar or mediastinal regions had no lymph node metastasis, with the size ranging from 0.4 to 2.5 cm (mean 1.1 cm 6 0.4). Among them, 34 (60.7%) regions had 18F-FDG–avid lymph nodes in the visual analysis, whereas only seven (12.5%) regions showed mild 99mTc-3PRGD2 accumulation (Fig 2). The T/B ratios of 18FFDG uptake were significantly higher than those of 99mTc-3PRGD2 accumulation (1.6 6 1.0 vs 1.0 6 0.1, P , .001). No significant correlation was observed between the T/B ratios of the two tracers in the benign lymph nodes (r = 20.02, P = .885). Both tracers had significantly higher T/B ratios in the metastatic lymph

Table 2 Receiver Operating Characteristic Curve Analysis of 99mTc-3PRGD2 SPECT/CT and 18F-FDG PET/CT in Diagnosis of Lung Cancer and Assessment of Lymph Node Metastasis, as Well as the Z Statistics Comparing the Two Methods Method Diagnosis of lung cancer 99m Tc-3PRGD2 SPECT/CT 18 F-FDG PET/CT Assessment of lymph node metastasis 99m Tc-3PRGD2 SPECT/CT 18 F-FDG PET/CT

Area Under the Curve

Standard Error

95% Confidence Interval

z Value

P Value .410 … … ,.001 … …

0.674 0.768

0.107 0.076

0.467, 0.881 0.620, 0.917

0.82 … … 8.0

0.970 0.792

0.009 0.035

0.952, 0.988 0.724, 0.859

… …

nodes than in the benign lymph nodes (P , .001). However, receiver operating characteristic analysis indicated that 99mTc-3PRGD2 SPECT/CT had a significantly higher area under curve than did 18F-FDG PET/CT in assessing lymph node involvement (0.970 [95% confidence interval: 0.952, 0.988] vs 0.792 [95% confidence interval: 0.724, 0.859]; z = 8.0, P , .001) (Table 2). The positive criteria is preferentially set as T/B ratio greater than 1.2 for 99mTc3PRGD2 SPECT/CT and T/B ratio greater than 1.6 for 18F-FDG PET/CT in the diagnosis of lymph node metastasis according to the region-based analysis. As shown in Table 3, the 99mTc-3PRGD2 SPECT/CT had significantly higher specificity and positive predictive value than did 18F-FDG PET/CT (94.6% vs 75.0%, P = .008 and 98.6% vs 94.1%, P = .010, respectively), whereas no significant difference was found between the other diagnostic values of the two methods, including the sensitivity (88.3% vs 90.7%, P = .557).

Correlation with Immunohistochemical Findings Twenty specimens were stained with integrin avb3, CD34, and Ki-67 to correlate with the imaging findings. In the 11 primary lung cancer specimens that underwent immunohistochemical analysis, 99mTc-3PRGD2

Table 3 Comparison of the Diagnostic Values of 99mTc-3PRGD2 SPECT/CT and 18F-FDG PET/CT in Diagnosis of Lung Cancer and Assessment of Lymph Node Metastasis Method and P Value Diagnosis of lung cancer (n = 79*) 99m Tc-3PRGD2 SPECT/CT 18 F-FDG PET/CT P value‡ Assessment of lymph node metastasis (n = 304*) 99m Tc-3PRGD2 SPECT/CT 18 F-FDG PET/CT P value‡

Positive Criteria

Sensitivity (%)

Specificity (%)

Accuracy (%)

PPV (%)

NPV (%)

T/B . 4·4 T/B . 8.0†

64.6 (42/65) 76.9 (50/65) .039

64.3 (9/14) 64.3 (9/14) .625

64.6 (51/79) 74.7 (59/79) .210

89.4 (42/47) 90.9 (50/55) .793

28.1 (9/32) 37.5 (9/24) .457

T/B . 1.2 T/B . 1.6†

88.3 (219/248) 90.7 (225/248) .557

94.6 (53/56) 75.0 (42/56) .008

89.4 (272/304) 87.8 (267/304) .362

98.6 (219/222) 94.1 (225/239) .010

64.6 (53/82) 64.6 (42/65) .998

Note.—PPV = positive predictive value, NPV = negative predictive value. * Lung lesions were calculated lesion by lesion and lymph nodes were calculated region by region for convenient correlation with the surgical records and pathologic reports. McNemar test was used to compare the diagnostic sensitivity, specificity, and accuracy. An x2 test was used to compare the other diagnostic values of the two methods. †

T/B ratios were used instead of standardized uptake value for better comparison with 99mTc-3PRGD2 SPECT/CT.

‡ For the difference between the two methods.

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Figure 2

Figure 2: Comparison of 18F-FDG PET/CT and 99mTc-3PRGD2 SPECT/CT in the evaluation of a lung adenocarcinoma with lymph node metastases and an inflammatory lung lesion with lymph node involvement. A, B, In a 45-year-old woman, maximum intensity projections show intense uptake of both 18F-FDG and 99m Tc-3PRGD2 in the right lower lung adenocarcinoma (black arrow) and the lymph node metastases (white arrow). The transaxial fusion images were shown in A, B, D, and E of Figure 3. C, D, In a 40-year-old women, as shown on the maximum intensity projections, the chronic inflammation in the right lower lung (black arrow) and the involved lymph nodes (white arrow) have intense 18F-FDG uptake but very low (black arrow) or no 99mTc-3PRGD2 accumulation.

accumulation was significantly correlated with integrin avb3 expression (r = 0.84, P = .001) and microvessel density counted on the CD34-stained slides (r = 0.63, P = .011), whereas 18 F-FDG uptake was positively correlated with Ki-67 index (r = 0.68, P = .021). However, moderate levels of integrin avb3 expression, microvessel density, and Ki-67 index were also observed in the granuloma (Fig 3). In comparison with the benign lymph nodes (n = 4), a significantly higher level of integrin avb3 expression was found in the metastasis (n = 4, P = .007), corresponding to the

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significantly higher accumulation of 99m Tc-3PRGD2 (P = .017). In contrast, no significant difference in 18F-FDG uptake was observed between the benign and metastatic lymph nodes (P = .286).

Discussion Tracers based on tripeptide sequence of RGD have been proved to have highbinding affinity to the integrin avb3 (8). Compared with the 18F-labeled RGDbased PET tracers previously translated to clinical use, such as 18F-Galacto-RGD (9), 18F-FPPRGD2 (10,11),


F-AH111585 (12), 18F-RGD-K5 (13), and 18F-alfatide (14), 99mTc-3PRGD2 holds advantages as an easy-labeling, cost-effective SPECT tracer by using the generator-produced 99mTc and broadly available SPECT system, rather than relying on an onsite cyclotron and the expensive PET system. Compared with other RGD-peptide SPECT tracers such as 99mTc-NC100692 (15), 99mTc3PRGD2 is a RGD dimer with three polyethylene glycol spacers to separate the functional structures, which has shown optimal biologic properties through a balance among tumor receptor binding, blood clearance kinetics, and biodistribution (16,17). Despite the lower resolution and sensitivity of SPECT than of PET, the present study demonstrated some interesting findings. First, 99mTc-3PRGD2 SPECT/CT is superior to 18F-FDG PET/CT in the diagnosis of lymph node metastasis (z = 8.0, P , .001). 99mTc-3PRGD2 SPECT/ CT reached a specificity of 94.6%, significantly higher than the 75.0% specificity of 18F-FDG PET/CT (P = .008), whereas no significant difference was found between the sensitivity of the two methods (88.3% vs 90.7%, P = .557). These findings indicate that the new method may hold a possibility to resolve the challenge in clinical decision making and surgical planning in patients with NSCLC, which usually needs mediastinoscopy or other invasive procedures (18,19). In the immunohistochemical analysis, the 18FFDG–avid lymph nodes with hyperplasia caused by acute or chronic inflammation had a very low expression of integrin avb3, corresponding well to the low accumulation of 99mTc-3PRGD2. In another study, we also found that 68GaNOTA-PRGD2, a PET tracer targeting the integrin avb3, did not accumulate in the 18F-FDG–avid inflammatory lymph nodes in rheumatoid arthritis patients (20). Therefore, integrin avb3 imaging may have an advantage over the 18FFDG metabolism imaging in identification of those NSCLC patients who can benefit from surgical resection or avoid unnecessary thoractomy through a precise lymph node staging. 963


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Figure 3

Figure 3: Comparison of 18F-FDG PET/CT, 99mTc-3PRGD2 SPECT/CT, and avb3-integrin immunohistochemical stains in lung adenocarcinoma with lymph node metastases and lung granuloma with lymph node inflammation. A–F, In a 45-year-old woman, the right lower lung adenocarcinoma (A–C) and mediastinal lymph node metastases (D–F) have intense 18F-FDG uptake, intense 99mTc-3PRGD2 accumulation, and high-level avb3-integrin expression. G–L, In a 53-year-old man, the right upper lung granuloma (G–I) shows intense 18F-FDG uptake, moderate 99mTc-3PRGD2 accumulation, and moderate-level avb3-integrin expression, whereas the mediastinal lymph nodes with inflammation (J–L) shows intense 18F-FDG uptake but without 99mTc-3PRGD2 accumulation and negative avb3integrin expression.

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Second, the malignant lung lesions, even as small as 1.1 cm, were all visually detectable on the 99mTc-3PRGD2 SPECT/CT images, despite the limited resolution of SPECT and significantly lower uptake of 99mTc-3PRGD2 than 18 F-FDG in the tumors. However, in contrast to the findings in lymph nodes, 99m Tc-3PRGD2 SPECT/CT did not show higher specificity than 18F-FDG PET/CT in the differentiation of lung lesions in this group of patients. The main reason may be the fact that six of the 12 benign cases enrolled in this study had granuloma, a benign disease also having moderately integrin avb3 expression (Fig 3). Third, 99mTc-3PRGD2 accumulation and 18F-FDG uptake were found in positive correlation in both primary lung cancer (P , .001) and lymph node metastases (P , .001), although different distribution of 99mTc-3PRGD2 and 18FFDG was found in some of the NSCLC tumors (Fig 1), indicating the distinctly different but closely related mechanism of the two tracers. However, there was no significant correlation between the two tracers in the benign lung lesions (P = .669) and lymph node hyperplasia (P = .885), indicating the existence of much complicated mechanisms in the benign lesions. In addition, 99mTc-3PRGD2 accumulation is significantly higher in patients with more metastases than in those with less or no metastasis, whereas 18F-FDG uptake had no such significant difference between the same groups of patients. These findings indicate that 99mTc-3PRGD2 accumulation may reflect the metastatic characteristic of NSCLC to some extent. The integrin avb3 is involved in the cell–cell and cell–matrix interactions and may play an essential role in tumor invasion and metastasis (3). A recent study reported that av-knockdown could strongly inhibit the migratory potentials and the metastatic ability of human prostate cancer cells (21). We have also found that the benign metastasizing leiomyoma in lungs showed high 99mTc-3PRGD2 accumulation but with minimal 18F-FDG uptake (22). Therefore, 99mTc-3PRGD2 may

have another advantage over 18F-FDG by helping predict the metastatic characteristic of tumor. Of this point, more data are needed to reach a definite conclusion. This study is limited by the relatively small number of benign cases and half of them had granuloma, a main cause of false-positive results in 18F-FDG PET/CT (23). Moreover, granuloma is also rich in angiogenesis and can express a moderate level of integrin avb3 receptor (Fig 3). Therefore, the diagnostic specificity for the lung lesions was relatively low for both 99m Tc-3PRGD2 SPECT/CT and 18FFDG PET/CT in this group of patients. Interestingly, the 18F-FDG–avid lymph nodes with acute or chronic inflammation showed a significantly lower 99m Tc-3PRGD2 accumulation, corresponding to a very low-level expression of integrin avb3, indicating a different integrin-related mechanism between the lymphoid follicular hyperplasia and granuloma. To our knowledge, this is the first report of the high specificity of integrin imaging in the diagnosis of lymph node metastasis from NSCLC. Further studies are warranted to clarify its value in guiding NSCLC surgical decision making and monitoring response to antiangiogenesis treatment as well (24). In conclusion, an imaging method targeting the integrin avb3 expression in NSCLC is established by using 99mTc3PRGD2 as the tracer, based on the broadly available, relatively low cost SPECT/CT technology. The high specificity of 99mTc-3PRGD2 SPECT/CT in the diagnosis of lymph node metastasis merits further clinical investigation of 99mTc-3PRGD2 as a tracer to expand the clinical application of SPECT/CT in oncology. Acknowledgments: The authors thank Haiqun Xing, BS, Tong Wang, BS, Yiming Liu, MD, and other related staff in Peking Union Medical College Hospital who have helped in the study performance and data collection. Disclosures of Conflicts of Interest: X.J. disclosed no relevant relationships. N.L. disclosed no relevant relationships. M.W. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed no relevant relationships.


Jin et al

Other relationships: a patent ZL200810239036.X issued. Y.M. disclosed no relevant relationships. B.J. disclosed no relevant relationships. X.S. disclosed no relevant relationships. S.L. disclosed no relevant relationships. J.L. disclosed no relevant relationships. Y.L. disclosed no relevant relationships. Q.C. disclosed no relevant relationships. K.Z. disclosed no relevant relationships. Z.L. disclosed no relevant relationships. J.S. disclosed no relevant relationships. F.L. disclosed no relevant relationships. F.W. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed no relevant relationships. Other relationships: has a patent ZL200810239036.X issued. Z.Z. disclosed no relevant relationships.

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