CT image, histogram distribution of CT attenuation value, and photomicrograph (hematoxylin-eosin stain; original magnification, X 40).

<p>(A) is a case of AIS, (B) is a case of MIA, (C) and (D) are cases of invasive adenocarcinoma. First three cases show pure GGNs without a solid component, whereas (D) shows GGN with 2 mm-solid component on CT image. As for histogram distribution, the vertical axis in each histogram shows the number of pixels in the segmented tumor. The red and blue lines indicate the values for 75^th and 97.5^th percentile. The horizontal axis shows the CT attenuation values. As compared with histograms of (A) AIS and (B) MIA, those of (B) MIA and (C) invasive adenocarcinoma show increased values in the 75^th and 97.5^th percentile. Tumor density was also increased, whereas tumor mass showed no difference. Histogram of MIA demonstrates a flat peak with high entropy as compared with that of AIS. Histogram graph of (D) shows two peaks, which is different from one peak of (A),(B), and (C). In a photomicrograph of (A) AIS, this circumscribed nonmucinous tumor grows purely with a lepidic pattern. No foci of invasion or scarring are seen. A photomicrograph of (B) MIA consists primarily of lepidic growth with a small (4.8 mm) upper area of acinar invasion. A photomicrograph of (C) invasive acinar adenocarcinoma consists of round to oval-shaped malignant glands invading a fibrous stroma 7 mm in length and a smaller area of lepidic growth only at the tumor periphery. Another photomicrograph of invasive acinar adenocarcinoma (D) shows centrally located bronchus, which is the main cause of solid component of CT image.</p

Receiver operating characteristic (ROC) curve for predicting invasive adenocarcinoma with imaging parameters.

<p>For invasive adenocarcinoma prediction, ROC curve based on the combination of the 75^th percentile CT attenuation value and entropy also shows significant diagnostic accuracy (AUC, 0.780).</p

Characteristics of lung adenocarcinoma with little solid component on CT in reference to invasion status (n = 191).

<p>Note—Classified According to the International Multidisciplinary Lung Adenocarcinoma Classification system.</p><p>Unless otherwise indicated, data are means ± standard deviation.</p><p>**Data number of individuals.</p><p>*<i>P</i><0.05</p><p>* <i>P</i> values were calculated with one-way ANOVA.</p><p>In terms of size variables and histogram analysis variables, <i>P</i> values are Bonferroni-corrected <i>P</i> values (Bonferroni-correction, <i>P</i><0.05÷2 for size variables and <i>P</i><0.05÷7 for histogram variables).</p><p><i>P</i>1 indicates the <i>P</i> values for post hoc analyses of AIS versus MIA.</p><p><i>P</i>2 indicates the <i>P</i> values for post hoc analyses of MIA versus invasive adenocarcinoma.</p><p><i>P</i>3 indicates the <i>P</i> values for post hoc analyses of AIS versus invasive adenocarcinoma.</p

DFS curves for AIS, MIA and invasive adenocarcinoma (IA) groups.

<p>DFS curves for AIS, MIA and invasive adenocarcinoma (IA) groups.</p

Multivariate analysis for stratification among AIS, MIA and invasive adenocarcinoma.

<p>Note.— <i>CI</i> confidence interval.</p><p>* <i>P</i><0.05.</p

Prevalence ratios (95% confidence intervals) for request of <i>EGFR</i> mutation testing by patient characteristics.

*<p>Multivariate analysis included 1,315 patients, and adjusted for gender, age at diagnosis, smoking status, mobility at admission, disease stage, tumor histology, and period of admission.</p

Correlation of imaging biomarker features with extent of invasion on pathology.

<p>Note—⁺Data are Spearman correlation coefficients.</p><p>* <i>P</i><0.05.</p><p>** <i>P</i><0.01.</p

Prevalence ratios (95% confidence intervals) for positive <i>EGFR</i> mutation status among 575 patients with available <i>EGFR</i> mutation data.

*<p>Multivariate analysis included 528 patients, and adjusted for gender, age at diagnosis, smoking status, and histology.</p

Epigenetic Inactivation of Heparan Sulfate (Glucosamine) 3-<i>O</i>-Sulfotransferase 2 in Lung Cancer and Its Role in Tumorigenesis

<div><p>Background</p><p>This study was aimed at investigating the functional significance of heparan sulfate (glucosamine) 3-<i>O</i>-sulfotransferase 2 (<i>HS3ST2</i>) hypermethylation in non-small cell lung cancer (NSCLC).</p> <p>Methodology/ Principal Findings </p><p><i>HS3ST2</i> hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using 298 formalin-fixed paraffin-embedded tissues and 26 fresh-frozen tissues from 324 NSCLC patients. MS-HRM (methylation-specific high-resolution melting) and EpiTYPER^TM assays showed substantial hypermethylation of CpG island at the promoter region of <i>HS3ST2</i> in six lung cancer cell lines. The silenced gene was demethylated and re-expressed by treatment with 5-aza-2′-deoxycytidine (5-Aza-dC). A promoter assay also showed the core promoter activity of HS3ST2 was regulated by methylation. Exogenous expression of HS3ST2 in lung cancer cells H460 and H23 inhibited cell migration, invasion, cell proliferation and whereas knockdown of HS3ST2 in NHBE cells induced cell migration, invasion, and cell proliferation <i>in</i><i>vitro</i>. A negative correlation was observed between mRNA and methylation levels of HS3ST2 in 26 fresh-frozen tumors tissues (ρ = -0.51, <i>P</i> = 0.009; Spearman’s rank correlation). <i>HS3ST2</i> hypermethylation was found in 95 (32%) of 298 primary NSCLCs. Patients with <i>HS3ST2</i> hypermethylation in 193 node-negative stage I-II NSCLCs with a median follow-up period of 5.8 years had poor overall survival (hazard ratio = 2.12, 95% confidence interval = 1.25–3.58, <i>P</i> = 0.005) compared to those without <i>HS3ST2</i> hypermethylation, after adjusting for age, sex, tumor size, adjuvant therapy, recurrence, and differentiation.</p> <p>Conclusions/ Significance</p><p>The present study suggests that <i>HS3ST2</i> hypermethylation may be an independent prognostic indicator for overall survival in node-negative stage I-II NSCLC.</p> </div

Knockdown of <i>HS3ST2</i> expression induced cell migration, cell invasion and cell proliferation.

<p>(A) HS3ST2 siRNA was transfected into NHBE cells, and siRNA-mediated knockdown was confirmed by Western blots. (B) Cell viability was measured by MTT assay determined by evaluating the absorbance of the converted dye at a wavelength of 570 nm. (C & D) The effect of HS3ST2 knockdown on cell migration (C) and invasion (D) was also measured in NHBE cells. NC indicates untreated normal control.</p