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Identification of Factors for the Preoperative Prediction of Tumour Subtype and Prognosis in Patients with T1 Lung Adenocarcinoma.

1. Introduction

The widespread use of computed tomography (CT) for screening lung cancers has made the detection of small peripheral pulmonary nodules possible [1]. Limited surgical resection has gradually gained acceptance for the treatment of such nodules and has excellent outcomes superior to those of lobectomy [2]. The most common histological type of lung cancer in recent years is adenocarcinoma [3]. The favourable prognosis of tumours containing larger areas of GGO appears to be independent of the tumour subtype and thus is potentially treatable via limited surgical resection [4-6]. However, because of the high frequency of lymph node involvement, use of this procedure is still controversial. Moreover, tumours <3 cm with both GGO areas and solid areas are often aggressive and invasive [7]. The lung adenocarcinoma subtypes have distinct GGO patterns [8] and thus can be readily identified via imaging; identification of the subtype is essential for selecting the appropriate surgical procedure for patients with small-sized lung cancers. Serum carcinoembryonic antigen (CEA) is also a useful diagnostic and prognostic factor for patients with lung cancer [9, 10]. Tomita et al. reported that lung adenocarcinomas with the nonlepidic dominant histologic subtype typically have high serum levels of CEA [11].

A better understanding of the molecular biology of lung adenocarcinoma might allow surgeons to better predict patient outcomes, as well as to define its subtypes. Our studies identified circulating microRNAs (miRNAs) as potential diagnostic biomarkers for early-stage lung cancer and suggest that several miRNAs can distinguish lung adenocarcinomas from squamous cell lung cancers [9, 12-14]. To our knowledge, only a few studies have shown that miRNAs can do so. Previous studies found that serum levels of the miRNA, miR-183, were higher in patients with lung cancer than in healthy individuals and that miR-183 promoted tumour cell growth and migration (i.e., acted as onco-miRNA) by targeting the transcription factor early growth response protein 1 [12, 15-17].

The combination of mRNA expression, miRNA expression, and DNA methylation has been used to identify prognostic classifiers for lung adenocarcinoma [18]; however, to our knowledge, no studies have evaluated the usefulness of combined CT findings, CEA levels, and miRNA expression. The aim of this study was to determine whether these parameters preoperatively predict prognosis, postoperative histological subtype, and lymph node involvement in patients with lung adenocarcinoma, toward the overall goal of identifying the subgroups that could benefit from limited resection. Our study was performed on patients with resected lung adenocarcinomas <30 mm.

2. Materials and Methods

2.1. Study Population. We retrospectively reviewed and analysed 87 consecutive patients (38 men, 49 women; mean age, 58 years; age range, 27-81 years) with pathological T1 lung adenocarcinoma (tumour size < 30 mm) who underwent surgical resection with curative intent at the Zhoushan Hospital (Zhejiang, China) between December 2011 and September 2014 and who were monitored for local recurrence and distant metastasis in follow-ups. Before surgery, all patients underwent routine or contrast-enhanced chest CT (Sensation 16; Siemens, Erlangen, Germany). The patients also underwent preoperative cardiopulmonary tests, abdominal CT or abdominal ultrasonography, brain magnetic resonance imaging or brain CT, and bone scanning. All patients underwent lobectomy with hilar and mediastinal lymphadenectomy or limited resection (segment or wedge) with lymph node sampling; none received preoperative chemotherapy or radiography. All specimens were formalin-fixed and stained with haematoxylin and eosin after surgery.

Non-small cell lung cancer (NSCLC) was diagnosed histologically by two pathologists in accordance with the 2004 World Health Organization criteria [19]. In accordance with the revised criteria of the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) [3], lung adenocarcinoma was classified as adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), or invasive adenocarcinoma (IAC). For IAC, there are five predominant growth patterns (lepidic, acinar, papillary, solid, and micropapillary) and four variants (invasive mucinous, colloid, foetal, and enteric) [20]. We also collected clinicopathological factors, including age, sex, lymphatic and vascular vessel invasion, bronchial invasion, preoperative serum CEA levels, and surgical procedures.

Blood was collected from all patients and 48 healthy individuals (normal controls) early in the morning. The serum was separated immediately after blood collection and stored at -80[degrees]C until use. The laboratory technicians were blinded to the patient's identity [17]. This study was approved by the Ethical Review Committee of the Zhoushan Municipal Government of China, and all patients provided written informed consent.

2.2. CT Imaging. High-resolution CT (HRCT) images were acquired by using a 16-row multislice CT scanner and the following parameters: 120 kVp and autoexposure control, 140-170 mA, 0.75-mm slice collimation, 12-mm feed/rotation, 0.5-second rotation time, and lung window settings (level = -500/width = 1500 HU). Moreover, all tumours were subsequently evaluated to estimate the extent of GGO via image reconstruction using a 1.0 mm slice thickness and a lung algorithm. In the current study, GGO was defined as the presence of misty increases in lung attenuation that did not obscure the bronchial or vascular walls, whereas solid tumours were tentatively defined as tumours in which the maximum diameter of consolidation relative to the maximum diameter of the tumour (consolidation/total tumour size) was >5 mm. Based on the diameters determined via HRCT, tumours were further classified into three groups: pure GGO (pGGO) (tumours without a solid component), mGGO (tumours with both GGO and solid components), and pure solid nodules (tumours with a solid component only) (Figure 1).

2.3. Serum Preparation, RNA Isolation, and Quantitative Reverse Transcription-Polymerase Chain Reaction. To obtain serum samples, 10 mL aliquots of peripheral blood were drawn into separate gel tubes and centrifuged within 30 min at 1,500 xg for 10 min at 4[degrees]C. The supernatants were transferred to 1.5 mL tubes and stored at -80[degrees]C until use. For detection of miRNA, RNA was isolated from 600 ^L serum samples using a mirVana PARIS RNA isolation kit (Applied Biosystems, Foster City, CA, USA) as specified by the manufacturer. RNA concentration was determined by using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Reverse transcription reactions were performed by using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was subsequently carried out on triplicate serum samples using TaqMan 2x Universal PCR Master Mix with no AmpErase UNG (Applied Biosystems) and an ABI 7500 Real-Time PCR system (Applied Biosystems). The qRT-PCR amplification conditions consisted of an initial cycle at 95[degrees]C for 10 min, followed by 40 cycles at 95[degrees]C for 15 s and 60[degrees]C for 1 min. Cycle threshold (Ct) values were calculated by using SDS 2.0.1 software (Applied Biosystems). No template controls were used at either the RT or PCR steps to ensure target-specific amplification. The control miRNA was U6 snRNA [21]. The 2- 1 method was used to calculate the average levels of serum miRNA (which were relative to the average levels of U6 snRNA) and to determine the fold change in the expression of the target miRNA relative to that of the miRNAs expressed in normal controls [22]. The mean Ct values, excluding outliers (i.e., replicates with Ct values differing by more than one cycle from the median), for miR-183 were calculated. In addition, if the average U6 Ct was not within 20 or 32 cycles, the assay was repeated at least once on some of the samples. Samples with low U6 snRNA levels were not included in the data analysis [17].

2.4. Follow-Up of the Patients. All patients were followed-up in our outpatient clinic at 3-month intervals for the first year and 6-month intervals thereafter. Tumour recurrence was assessed via CT and by measuring serum CEA levels. The length of the follow-up period ranged from 2 to 46 months, with a mean of 29 months and a median of 32 months. The last follow-up was conducted in June 2015. The endpoint of this study was the death of the patient or was censored at the time of the last follow-up. Overall survival was defined as the time (in months) from surgery to either death or the last follow-up.

2.5. Statistical Analysis. Statistical analyses were performed by using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA) and MedCalc 9.0 software (MedCalc Software Inc., Mariakerke, Belgium). The data were examined according to the degree of homogeneity. The unpaired t-test or Mann-Whitney U test was used to analyse the differences between patients and controls and the correlations between miRNA expression levels and the clinicopathological features of the patients. Receiver operating characteristic (ROC) curves were generated to assess the diagnostic accuracy of each parameter. The areas under the curves (AUCs) were calculated and compared with each other via a nonparametric approach.

The five factors (age, serum CEA level, serum miR-183 level, tumour size, and nodule type) that differed significantly between patients with AIS or MIA and those with IAC were combined to predict AIS and MIA versus IAC; multiple logistic regression analysis and MedCalc 9.0 software were used to calculate the predictive value. Multiple logistic regression analysis was also performed to assess the diagnostic accuracy of total serum miRNA levels in the lung adenocarcinoma patients. All statistical tests were two-sided, and a p value < 0.05 was considered statistically significant.

3. Results

The characteristics of the patients and tumours are summarized in Table 1. The tumours were stratified into two groups according to their pathological characteristics: AIS or MIA (41 patients, 47.1%) and IAC (46 patients, 52.9%) (Figure 1). Neither lymph node metastasis nor visceral pleural invasion was observed in patients with AIS, MIA, or lepidic-predominant IAC. Of the four patients with lymph node metastasis, three had papillary-predominant IAC and one had solid-predominant IAC. Pure solid nodules were also not observed in the patients with AIS or MIA. One of the 23 patients with papillary-predominant IAC presented with pGGO. Preoperative radiological findings, serum levels of CEA, serum levels of miR-183, tumour size, and visceral pleural invasion status differed significantly between patients with AIS or MIA and those with IAC (p: <0.001, 0.015, 0.042, <0.001, and 0.003, resp.) (Table 2). Median age was higher in the IAC group than in the AIS and MIA group (p < 0.001).

Factors that predict the IAC subtype versus the AIS and MIA subtypes were identified by using a logistic regression model. In the univariate analysis, older age, the presence of pure solid nodules, a high level of CEA, and tumour size >20 mm significantly predicted the IAC subtype (p: 0.001, <0.001, 0.024, and <0.001, resp.) (Table 3). In the multivariate analysis, the presence of pure solid nodules and tumour size >20 mm were independent predictors of the IAC subtype (p: 0.017 and <0.001, resp.). ROC curves were generated to assess the IAC prediction accuracy of the four factors identified in the univariate analysis, as well as miR-183. Figure 2 shows the true-positive ratios (sensitivity) and false-positive ratios (1 minus specificity) for these factors. The AUCs for age, serum CEA, miR-183, tumour size, and nodule type were, respectively, 0.743, 0.652, 0.627, 0.915, and 0.812 (p < 0.001), with a sensitivity of 71.74%, 60.87%, 52.17%, 82.61%, and 97.83% (p = 0.001) and a specificity of 70.73%, 75.61%, 70.73%, 92.68%, and 43.90% (p < 0.001). We also assessed the IAC prediction accuracy of these five factors in combination via multivariate logistic regression analysis. The AUC of the five-factor combination was 0.946, with a sensitivity of 89.13% and a specificity of 95.12% (p < 0.001).

To further distinguish IAC from AIS and MIA, we performed univariate and multivariate analyses using the optimal cut-off values calculated from the ROC curves. These were 57 years, 2.12ng/mL, 1.233 ([2.sup.-[DELTA][DELTA]Ct]), 11mm, 1, and 0.499 for age, serum CEA, miR-183, tumour size, pGGO or mGGO versus pure solid nodules, and the five-factor combination, respectively (Table 4). According to the univariate analysis, patients were more likely to be diagnosed with IAC than with AIS or MIA if they were >57 years of age or had a pure solid nodule, a tumour size >11 mm, a serum CEA level >2.12 ng/mL, or a miR-183 level >1.233 ([2.sup.-[DELTA][DELTA]Ct) (p: <0.001, 0.001, <0.001, 0.001, and 0.020, resp.). Using the cut-off value of 0.499, the combination of these five factors also indicated a high possibility of IAC and was an independent diagnostic factor for IAC in the multivariate analysis (p < 0.001).

Additional studies assessed the overall survival of patients with lung adenocarcinoma according to the preoperative factors identified above using the optimal cut-off values. The results of these studies showed that patients with > 1 pure solid nodule, a tumour >11 mm, a serum CEA level >2.12 ng/mL, or a predictive value >0.499 had shorter overall survival times than their respective counterparts (p: 0.0002, 0.035, 0.025, and 0.042, resp.) (Figure 3). However, there were no significant differences between patients >57 and [less than or equal to] 57 years of age (p = 0.874) or with a serum miR-183 level >1.233 and [less than or equal to] 1.233 ([2.sup.-[DELTA][DELTA]Ct]) (p = 0.854).

4. Discussion

This study shows that specific preoperative factors can predict the pathological subtype of T1 lung adenocarcinomas. The five-factor combination of age, nodule type, serum CEA level, serum miR-183 level, and tumour size had an AUC of 0.946, with a sensitivity of 89.13% and a specificity of 95.12%. Moreover, based on optimal cut-off values, a tumour size >11 mm and a serum CEA level >2.12 ng/mL predicted the prognosis of lung adenocarcinoma patients, as did the presence of a pure solid nodule and a value >0.499 for the five-factor combination.

The IASLC/ATS/ERS classification system clearly presents the prognosis values for the predominant pathological subtypes and categorizes tumour behaviour according to the genetic diversity of the subtypes [23-25]. Two studies showed that lung adenocarcinoma patients with AIS or MIA but no lymph node metastasis had a nearly 100% overall survival rate, whereas those with histologic acinar-, papillary-, solid, or micropapillary-predominant IAC had relatively poor prognosis [25,26]. Limited resection has been recently advocated for patients with AIS or MIA because it preserves lung function [27] but is not suitable for patients with IAC. Thus, using preoperative factors to predict the histologic subtype of lung adenocarcinomas would be helpful for determining the appropriate surgical procedure.

Because preoperative diagnosis of AIS and MIA via transbronchial biopsy or percutaneous needle biopsy is difficult, measurement of circulating tumour marker levels and CT imaging, which is a noninvasive procedure, are attractive alternatives. Image analysis via high-resolution chest CT provides the most useful information for identifying AIS and MIA [6]. Our results showed that pGGO was closely associated with AIS and MIA in most instances (as the exception, one patient with a pGGO tumour presented with papillary-predominant IAC). In a recent study, only 12% of cases with pGGO were ultimately diagnosed as IAC, most of which were papillary-predominant [28]. In our study, pure solid nodules were associated with IAC but not with AIS or MIA, and there was no obvious association of mGGO with AIS, MIA, or IAC. The presence of a pure solid nodule independently predicted IAC with an AUC of 0.812, and patients with a pure solid nodule had a worse survival rate than did those with a pGGO or mGGO tumour. In agreement with our results, several studies found that the proportion of GGO areas relative to the total tumour area strongly predicted overall survival in patients with clinical T1N0M0 lung adenocarcinoma [29,30]. Although our study shows that GGO is useful for diagnosing and predicting the prognosis of the adenocarcinoma subtypes, other factors should be identified to improve sensitivity and specificity, especially for mGGO tumours owing to their high recurrence rate [29].

The proposed eighth edition of the tumour, node, and metastasis (TNM) classification system for lung cancer suggests revisions that might improve lung cancer diagnosis and potentially subcategorize T1 tumours as T1a (<10 mm), T1b (>10 to [less than or equal to] 20mm), or T1c (>20 to [less than or equal to] 30 mm) [31]. We found that tumour size was an independent diagnostic factor for IAC that remarkably distinguished IAC from AIS and MIA when an optimal cut-off value of 11 mm was used. Mao et al. reported that 8.9 mm was the optimal tumour size cutoff value for differentiating between preinvasive (atypical adenomatous hyperplasia and AIS) and invasive (MIA and IAC) lesions [32]. However, Lee et al. recommended optimal cut-off values of 10 mm and 14 mm for distinguishing preinvasive lesions from invasive pulmonary lesions in cases of pGGO and partly sGGO, respectively [33]. The appropriate value might differ according to the size of the lung window and patient cohort, which would be determined by the institute. Larger tumour size significantly correlated with a higher percentage of metastatic N2 lymph nodes and poor prognosis in patients with T1 lung adenocarcinoma [34]. Our results also demonstrated that a tumour size >11 mm predicted poor overall survival. Further surgical intervention and radiotherapy or chemotherapy should be indicated for such tumours.

Serum CEA is a useful circulating biomarker and prognostic factor for lung cancer. However, the optimal cutoff value for serum CEA level varies in the literature [7, 9]. Using the optimal cut-off value identified in our study (>2.12 ng/mL), we found that serum CEA level was associated not only with IAC, but also with poor overall survival in T1 lung adenocarcinoma patients [11, 35]. Several studies have shown a correlation between high preoperative serum CEA levels and poor survival after lung cancer resection, which is consistent with our results [7,11, 36].

Circulating miRNAs were identified as novel, noninvasively detected biomarkers for diagnosing and predicting the prognosis of lung cancers [9, 12, 37, 38]. Herein, we are the first to report that elevated levels of the onco-miRNA, miR-183, were closely associated with IAC and could distinguish IAC from AIS and MIA when an optimal cut-off value of 1.2328 (AUC = 0.627) was used. Previous studies showed increased circulating levels of miR-183 in several cancer types and suggested that miR-183 was a potential biomarker for the diagnosis of carcinomas including lung carcinomas [12,17,39, 40].

Nevertheless, it was sometimes difficult to accurately predict the lung adenocarcinoma subtype and thus the surgical procedure that would be most successful, using only a single factor. To potentially improve accuracy, we combined the five factors (age, nodule type, serum CEA level, serum miR-183 level, and tumour size) that significantly differentiated AIS and MIA from IAC preoperatively. This resulted in a cutoff value (>0.499) that further increased diagnostic accuracy (AUC, 0.946; 89.1% sensitivity, and 95.1% specificity) and adequately excluded IAC. The five-factor combination represents a useful means of selecting patients suitable for limited resection, and it was remarkably associated with poor prognosis in our study. Therefore, we successfully validated the use of limited resection by using the following parameters: age [less than or equal to] 57 years, serum CEA level [less than or equal to] 2.12 ng/mL, serum miR-183 level [less than or equal to] 1.233 ([2.sup.-[DELTA][DELTA]Ct]), tumour size [less than or equal to] 11 mm, and the absence of a pure solid nodule.

In conclusion, our study highlights the potential usefulness of radiological findings, serum CEA and miR-183 levels, and tumour size in predicting the subtype and prognosis of lung adenocarcinomas. The five-factor combination allows clinicians to distinguish AIS and MIA from IAC and to predict the prognosis of lung adenocarcinomas for which limited surgical resection or adjusted therapy is appropriate. Further studies with more patients and longer follow-up periods after limited resection are recommended.

http://dx.doi.org/10.1155/2016/9354680

Competing Interests

The authors declare that they have no competing interests.

Authors' Contributions

Wang-Yu Zhu and Yong-Kui Zhang contributed equally to this work.

Acknowledgments

This research was supported in part by grants from the Science and Technology Bureau of Zhoushan (nos. 2011C12039, 2011C12040, and 2014C31063) to Yong-Kui Zhang, WangYu Zhu, and Zhi-jun Chen; the Medical Bureau of Zhejiang Province (nos. 2015ZDA032, 2016RCB020) to Han-bo Le and Wang-Yu Zhu; the Science Technology Department of Zhejiang Province (no. 2016C37008) to Xiao-fei Hu; the Zhejiang Provincial Natural Science Foundation of China (no. LQ17H160001) to Wang-Yu Zhu; and the Health Ministry-Medical Bureau of Zhejiang Province (no. WKJ2014-2-021) to Yong-Kui Zhang. The funders had no role in study design, data collection or analysis, the decision to publish, or the preparation of the manuscript.

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Wang-Yu Zhu, (1,2) Yong-Kui Zhang, (2,3) Zhen-da Chai, (2,3) Xiao-fei Hu, (4) Lin-lin Tan, (2,3) Zhao-yu Wang, (2) Zhi-jun Chen, (2,3) and Han-bo Le (2,3)

(1) Laboratory of Cytobiology and Molecular Biology, Zhoushan Hospital of Wenzhou Medical University, Zhoushan, Zhejiang 316021, China

(2) Lung Cancer Research Centre, Zhoushan Hospital of Wenzhou Medical University, Zhoushan, Zhejiang 316021, China

(3) Department of Cardio-Thoracic Surgery, Zhoushan Hospital of Wenzhou Medical University, Zhoushan, Zhejiang 316021, China

(4) Department of Science Education, Zhoushan Hospital of Wenzhou Medical University, Zhoushan, Zhejiang 316021, China

Correspondence should be addressed to Han-bo Le; zslehanbo@163.com

Received 22 July 2016; Revised 26 October 2016; Accepted 15 November 2016

Academic Editor: Stamatios E. Theocharis

Caption: Figure 1: Radiological and histopathological findings for the different subtypes of lung adenocarcinoma. (a) High-resolution computed tomography (HRCT) scan of an adenocarcinoma in situ 8 mm in diameter with a radiologically pure ground-glass opacity (GGO) nodule. (b) Microscopic examination of the tumour in (a). (c) HRCT scan of a minimally invasive adenocarcinoma 16 mm in diameter with a radiologically mixed GGO nodule. (d) Microscopic examination of the tumour in (c). (e) HRCT scan of a papillary-predominant invasive adenocarcinoma 13 mm in diameter with a radiologically mixed GGO nodule. (f) Microscopic examination of the tumour in (e). All photographs show haematoxylin-eosin staining; original magnification x400.

Caption: Figure 2: Receiver operating characteristic curves assessing the accuracy of several factors in predicting invasive adenocarcinoma (IAC) (41 patients) versus adenocarcinoma in situ or minimally invasive adenocarcinoma (41 patients). The p values for age (a), serum carcinoembryonic antigen (CEA) level (b), microRNA-183 (miR-183) level (c), tumour size (d), nodule type (e), and the combination of these five factors (f) as determined via logistic regression were <0.001, 0.010, 0.035, <0.0001, <0.001, and <0.001, respectively. AUC, area under the curve; pGGO, pure ground-glass opacity; mGGO, mixed ground-glass opacity.

Caption: Figure 3: Kaplan-Meier analysis of overall survival in patients with lung adenocarcinoma. Overall survival was stratified according to nodule type (a) and the optimal cut-off values for tumour size (b), serum carcinoembryonic antigen (CEA) level (c), and the five-factor combination (d).
Table 1: Characteristics of the patients and tumours.

Variable         Category                               Number (%)

Sex              Male                                   38 (43.7)
                 Female                                 49 (56.3)
Median age                                         58.0 [+ or -] 11.1
(range)                                                  (27-81)

Smoking          Current smoker                         66 (75.9)
                 Never smoked                           21 (24.1)
                 Pure GGO                               19 (21.8)
Radiological     Mixed GGO                              51 (58.6)
finding          Pure solid nodule                      17 (19.6)

CEA              [less than or equal to] 5              74 (85.1)
                 ng/mL >5 ng/mL                         13 (14.9)

Serum            [less than or equal to] 1.0            43 (49.4)
miR-183          ([2.sup.-[DELTA][DELTA]Ct)             44 (50.6)
                 >1.0                                   42 (48.3)
                 (2([2.sup.-[DELTA][DELTA]Ct)
                 [less than or equal to] 10 mm

Tumour           >10 to [less than or equal             30 (34.5)
size             to] 20 mm
                 >21 to [less than or equal             15 (17.2)
                 to] 30 mm
Nodule           Single                                 82 (94.3)
number           Multiple                                5 (5.7)
                 AIS                                    19 (21.8)
                 MIA                                    22 (25.3)

Predominant      Lepidic-predominant IAC                11 (12.7)
subtype          Papillary-predominant IAC              23 (26.4)
                 Acinar-predominant IAC                  9 (10.3)
                 Solid-predominant IAC                   2 (2.3)
                 Invasive mucinous IAC                   1 (1.2)

Lymph node       Present                                 4 (4.6)
metastasis

Visceral         Present                                 9 (10.3)
pleural          0                                       5 (5.7)
invasion

Pathological     I                                      76 (87.4)
stage            II                                      4 (4.6)
                 III                                     2 (2.3)

CEA, carcinoembryonic antigen; GGO, ground-glass opacity; AIS,
adenocarcinoma in situ; MIA, minimally invasive adenocarcinoma; IAC,
invasive adenocarcinoma.

Table 2: Correlation of the resected lung adenocarcinoma
classification (n = 87) with clinical characteristics.

Variable                  Category         AIS and MIA (n = 41)

Sex                         Male                15 (36.6)
                           Female               26 (63.4)
Median age (years)                          52.8 [+ or -] 10.3

Smoking                 Always smoked           33 (80.5)
                        Never smoked             8 (19.5)
                          Pure GGO              18 (43.9)
Radiological              Mixed GGO             23 (56.1)
finding               Pure solid nodule             0

CEA [IQR                                     1.17-2.32 (1.78)
(median)]
(ng/mL)

Serum miR-183                               0.232-1.63 (0.790)
[IQR (median)]             <10 mm               36 (87.8)

Tumour size          [less than or equal         4 (9.8)
                     to] 10 mm to [less
                     than or equal to]
                            20 mm
                     >21 to [less than]          1 (2.4)
                     or equal to] 30 mm

Lymph node                 Absent               41 (100.0)
metastasis                 Present                  0

Visceral                   Absent               41 (100.0)
pleural                    Present                  0
invasion

Pathological                0/IA                40 (97.6)
stage                  IB/IIA/IIB/IIIA           1 (2.4)

Variable                IAC (n = 46)        P value

Sex                      23 (50.0)           0.279
                         23 (50.0)
Median age (years)   62.7 [+ or -] 9.8     <0.001 *

Smoking                  33 (71.7)        0.453 0.453
                         13 (28.3)
                          1 (2.2)
Radiological             28 (60.9)         <0.001 *
finding                  17 (36.9)

CEA [IQR              1.48-4.31 (2.30)      0.015 *
(median)]
(ng/mL)

Serum miR-183        0.684-4.33 (1.325)     0.042 *
[IQR (median)]            6 (13.0)

Tumour size              26 (56.5)         <0.001 *

                         14 (30.5)

Lymph node               42 (91.3)           0.119
metastasis                4 (8.7)

Visceral                 37 (80.4)          0.003 *
pleural                   9 (19.6)
invasion

Pathological             30 (65.2)         <0.001 *
stage                    16 (34.8)

* Statistically significant p value

CEA, carcinoembryonic antigen; GGO, ground-glass opacity; IQR,
interquartile range; AIS, adenocarcinoma in situ; MIA, minimally
invasive adenocarcinoma; IAC, invasive adenocarcinoma.

Table 3: Univariate and multivariate analysis to predict
pathological subtypes.

Variable                                 Univariate

                                  OR        95% CI       p value

Age >60 years                    4.994   2.005-12.438    0.001 *
Sex: male                        0.577   0.244-1.362     0.209
Smoking status: current          1.625   0.595-4.436     0.343
Pure solid nodule on            36.561   4.972-268.878   <0.001 *
  radiology
Serum CEA level                 6.129    1.270-29.583    0.024 *
  >5 ng/mL
Serum miR-183 level >1.0        2.006     0.853-4.716     0.110
  ([2.sup.-[DELTA][DELTA]Ct)

Tumour size >21 to [less        21.981   6.988-69.142    <0.001 *
than or equal to] 30 mm

Variable                                 Multivariate

                                  OR        95% CI       p value

Age >60 years                   2.167     0.547-8.593     0.271
Sex: male                         --          --            --
Smoking status: current           --          --            --
Pure solid nodule on            12.681   1.562-102.972   0.017 *
  radiology
Serum CEA level                 1.143    0.117-11.131     0.908
  >5 ng/mL
Serum miR-183 level >1.0          --          --            --
  ([2.sup.-[DELTA][DELTA]Ct)

Tumour size >21 to [less        9.609    2.882-32.038    <0.001 *
than or equal to] 30 mm

* Statistically significant p value; -, not included in the
multivariate analysis OR, odds ratio; CEA, carcinoembryonic antigen;
CI, confidence interval.

Table 4: Univariate and multivariate analysis to predict pathological
subtype using optimal cut-off values.

Variable                          Cut-off value
                                                               OR

Age                                 >57 years                 6.135
Radiological findings          >1 pure solid nodule          35.217
Serum CEA level                    >2.12 ng/mL                4.822
Serum miR-183 level     >1.233 ([2.sup.-[DELTA][DELTA]Ct)     2.900
Tumour size                           >11 mm                 60.167
Combined five factors                 >0.499                 159.900

Variable                  Univariate
                            95% CI       P value      OR

Age                      2.421-15.545    <0.001 *   1.307
Radiological findings   4.420-280.601    0.001 *    3.304
Serum CEA level          1.909-12.181    0.001 *    0.957
Serum miR-183 level      1.183-7.112     0.020 *    0.428
Tumour size             14.822-244.236   <0.001 *   3.106
Combined five factors   29.287-873.024   <0.001 *   60.033

Variable                 Multivariate
                            95% CI       P value

Age                      0.209-8.175      0.774
Radiological findings    0.317-34.440     0.318
Serum CEA level          0.158-5.788      0.961
Serum miR-183 level      0.046-4.006      0.457
Tumour size              0.253-38.068     0.375
Combined five factors   2.462-1463.959   0.012 *

* Statistically significant p value

OR, odds ratio; CI, confidence interval; mGGO, mixed ground-glass
opacity; CEA, carcinoembryonic antigen.
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Title Annotation:Research Article
Author:Zhu, Wang-Yu; Zhang, Yong-Kui; Chai, Zhen-da; Hu, Xiao-fei; Tan, Lin-lin; Wang, Zhao-yu; Chen, Zhi-j
Publication:Disease Markers
Article Type:Report
Date:Jan 1, 2017
Words:6336
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