Exfoliate cancer cell analysis in rectal cancer surgery: comparison of laparoscopic and transanal total mesorectal excision, a pilot study.

PURPOSE: Minimally invasive surgery (MIS) is currently the standard treatment for rectal cancer. However, its limitations include complications and incomplete total mesorectal resection (TME) due to anatomical features and technical difficulties. Transanal TME (TaTME) has been practiced since 2010 to improve this, but there is a risk of local recurrence and intra-abdominal contamination. We aimed to analyze samples obtained through lavage to compare laparoscopic TME (LapTME) and TaTME. METHODS: From June 2020 to January 2021, 20 patients with rectal cancer undergoing MIS were consecutively and prospectively recruited. Samples were collected at the start of surgery, immediately after TME, and after irrigation. The samples were analyzed for carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) through a quantitative real-time polymerase chain reaction. The primary outcome was to compare the detected amounts of CEA and CK20 immediately after TME between the surgical methods. RESULTS: Among the 20 patients, 13 underwent LapTME and 7 underwent TaTME. Tumor location was lower in TaTME (7.3 cm vs. 4.6 cm, P=0.012), and negative mesorectal fascia (MRF) was more in LapTME (76.9% vs. 28.6%, P=0.044). CEA and CK20 levels were high in 3 patients (42.9%) only in TaTME. There was 1 case of T4 with incomplete purse-string suture and 1 case of positive MRF with dissection failure. All patients were followed up for an average of 32.5 months without local recurrence. CONCLUSION: CEA and CK20 levels were high only in TaTME and were related to tumor factors or intraoperative events. However, whether the detection amount is clinically related to local recurrence remains unclear.

Molecular Subtypes and Tumor Microenvironment Characteristics of Small-Cell Lung Cancer Associated with Platinum-Resistance.

Although molecular subtypes of small-cell lung cancer (SCLC) have been proposed, their clinical relevance and therapeutic implications are not fully understood. Thus, we aimed to refine molecular subtypes and to uncover therapeutic targets. We classified the subtypes based on gene expression (n = 81) and validated them in our samples (n = 87). Non-SCLC samples were compared with SCLC subtypes to identify the early development stage of SCLC. Single-cell transcriptome analysis was applied to dissect the TME of bulk samples. Finally, to overcome platinum resistance, we performed drug screening of patient-derived cells and cell lines. Four subtypes were identified: the ASCL1+ (SCLC-A) subtype identified as TP53/RB-mutated non-SCLC representing the early development stage of SCLC; the immune activation (SCLC-I) subtype, showing high CD8+/PD-L1+ T-cell infiltration and endothelial-to-mesenchymal transition (EndMT); the NEUROD1 (SCLC-N) subtype, which showed neurotransmission process; and the POU2F3+ (SCLC-P) subtype with epithelial-to-mesenchymal transition (EMT). EndMT was associated with the worst prognosis. While SCLC-A/N exhibited platinum sensitivity, the EndMT signal of SCLC-I conferred platinum resistance. A BET inhibitor suppressed the aggressive angiogenesis phenotype of SCLC-I. We revealed that EndMT development contributed to a poor outcome in SCLC-I. Moreover, heterogenous TME development facilitated platinum resistance. BET inhibitors are novel candidates for overcoming platinum resistance.

A host non-coding RNA, nc886, plays a pro-viral role by promoting virus trafficking to the nucleus.

Elucidation of the interplay between viruses and host cells is crucial for taming viruses to benefit human health. Cancer therapy using adenovirus, called oncolytic virotherapy, is a promising treatment option but is not robust in all patients. In addition, inefficient replication of human adenovirus in mouse hampered the development of an in vivo model for preclinical evaluation of therapeutically engineered adenovirus. nc886 is a human non-coding RNA that suppresses Protein Kinase R (PKR), an antiviral protein. In this study, we have found that nc886 greatly promotes adenoviral gene expression and replication. Remarkably, the stimulatory effect of nc886 is not dependent on its function to inhibit PKR. Rather, nc886 facilitates the nuclear entry of adenovirus via modulating the kinesin pathway. nc886 is not conserved in mouse and, when xenogeneically expressed in mouse cells, promotes adenovirus replication. Our investigation has discovered a novel mechanism of how a host ncRNA plays a pro-adenoviral role. Given that nc886 expression is silenced in a subset of cancer cells, our study highlights that oncolytic virotherapy might be inefficient in those cells. Furthermore, our findings open future possibilities of harnessing nc886 to improve the efficacy of oncolytic adenovirus and to construct nc886-expressing transgenic mice as an animal model.

Anti-tumor activity of BET inhibitors in androgen-receptor-expressing triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease comprising several subtypes. Androgen-receptor (AR) signaling has been targeted by several investigational agents in luminal AR subtype TNBCs. Bromodomain (BRD) and extra-terminal motif (BET) protein inhibitors have been shown to attenuate AR signaling in metastatic castration-resistant prostate cancer and to overcome enzalutamide resistance. We demonstrated potent anti-tumor effects of the BET inhibitor JQ1 against AR-positive TNBC cell lines using cell viability and cell cycle analysis. To reveal the mechanisms of JQ1 effects, multiplex gene expression analysis and immunoblotting assays were used. We examined in vivo effects of JQ1 in a xenograft model of AR expressing TNBC. JQ1 exhibited its anti-proliferative activity by inducing apoptosis and cell cycle arrest. JQ1 activity was not mediated by MYC downregulation. Instead, JQ1 blocked the interactions among the ATPase-family AAA-domain-containing 2 protein (ATAD2), BRD2, BRD4, and AR; effectively suppressing the expression of AR associated targets. In addition, JQ1 showed significant anti-tumor activity in vivo in TNBC xenograft mouse models as a monotherapy and in combination with anti-AR therapy. Taken together, our results showed that the BET inhibitor JQ1 is a promising therapeutic agent for the treatment of AR-positive TNBC.

Author Correction: nc886 is induced by TGF-ß and suppresses the microRNA pathway in ovarian cancer.

In the original version of the Supplementary Information file associated with this Article, Supplementary Fig. 18 panel b was inadvertently replaced with a duplicate of panel a. The error has now been fixed and the corrected version of the Supplementary Information PDF is available to download from the HTML version of the Article.

nc886 is induced by TGF-ß and suppresses the microRNA pathway in ovarian cancer.

Transforming growth factor-ß (TGF-ß) signaling and microRNAs (miRNAs) are important gene regulatory components in cancer. Usually in advanced malignant stages, TGF-ß signaling is elevated but global miRNA expression is suppressed. Such a gene expression signature is well illustrated in a fibrosis (or mesenchymal) subtype of ovarian cancer (OC) that is of poor prognosis. However, the interplay between the two pathways in the OC subtype has not yet been elucidated. nc886 is a recently identified non-coding RNA implicated in several malignancies. The high expression of nc886 is associated with poor prognosis in 285 OC patients. Herein, we find that in OC nc886 expression is induced by TGF-ß and that nc886 binds to Dicer to inhibit miRNA maturation. By preventing the miRNA pathway, nc886 emulates TGF-ß in gene expression patterns and potentiates cell adhesion, migration, invasion, and drug resistance. Here we report nc886 to be a molecular link between the TGF-ß and miRNA pathways.

Why do consumers respond to eco-labels? The case of Korea.

Although eco-labels were introduced with the intention of encouraging eco-friendly purchasing behavior by consumers, they have had little effect on consumers' purchasing decisions, and therefore a significant gap exists between eco-label awareness and actual purchasing behavior. The aim of this study was to analyze consumer preference, in terms of public and private values, for two types of Korean eco-label that have been administered by the Korean government since 1992. Analyses were based on a structural equation model, employing the theory of reasoned action. Data were collected by survey. The results indicate that although general consumers are highly aware of the publicly valuable information that eco-labels provide, privately valuable information exerts far greater power over their purchasing intentions. Therefore, a supplementary policy that converts public value to private value could promote the purchase of eco-labeled products.

Oncogenic function and clinical implications of SLC3A2-NRG1 fusion in invasive mucinous adenocarcinoma of the lung.

The neuregulin 1 (NRG1) fusion is a recently identified novel driver oncogene in invasive mucinous adenocarcinoma of the lung (IMA). After identification of a case of SLC3A2-NRG1 in a patient with IMA, we verified this fusion gene in a cohort of 59 patients with IMA. Targeted cancer panel sequencing and RT-PCR identified the possible coexistence of other driver oncogenes. Among 59 IMAs, we found 16 NRG1 fusions (13 SLC3A2-NRG1 and 3 CD74-NRG1). Of 16 patients with NRG1 fusions, concurrent KRAS codon 12 mutations were found in 10 cases. We also found concurrent NRAS Q61L mutation and EML4-ALK fusion in additional two cases with NRG1 fusions. When comparing overall survival (OS) according to the presence of NRG1 fusions showed that patients harboring NRG1 fusions had significantly inferior OS than those without NRG1 fusions (hazard ratio = 0.286; 95% confidence interval, .094 to .865). Ectopic expression of the SLC3A2-NRG1 fusion in lung cancer cells increased cell migration, proliferation and tumor growth in vitro and in xenograft models, suggesting oncogenic function for the fusion protein. We found that the SLC3A2-NRG1 fusion promoted ERBB2-ERBB3 phosphorylation and heteroduplex formation and activated the downstream PI3K/AKT/mTOR pathway through paracrine signaling. These findings suggested that the SLC3A2-NRG1 fusion was a driver in IMA with an important prognostic impact. SLC3A2-NRG1 should be considered a therapeutic target for patients with IMA.

nc886, a non-coding RNA and suppressor of PKR, exerts an oncogenic function in thyroid cancer.

nc886 is a recently identified cellular non-coding RNA and its depletion leads to acute cell death via PKR (Protein Kinase RNA-activated) activation. nc886 expression is increased in some malignancies, but silenced in others. However, the precise role of nc886/PKR is controversial: is it a tumor suppressor or an oncogene? In this study, we have clarified the role of nc886 in thyroid cancer by sequentially generating PKR knockout (KO) and PKR/nc886 double KO cell lines from Nthy-ori 3-1, a partially transformed thyroid cell line. Compared to the wildtype, PKR KO alone does not exhibit any significant phenotypic changes. However, nc886 KO cells are less proliferative, migratory, and invasive than their parental PKR KO cells. Importantly, the requirement of nc886 in tumor phenotypes is totally independent of PKR. In our microarray data, nc886 KO suppresses some genes whose elevated expression is associated with poor survival confirmed by data from total of 505 thyroid cancer patients in the Caner Genome Atlas project. Also, the nc886 expression level tends to be elevated and in more aggressively metastatic tumor specimens from thyroid cancer patients. In summary, we have discovered nc886's tumor-promoting role in thyroid cancer which has been concealed by the PKR-mediated acute cell death.

Relationship between insulin-like growth factor axis gene polymorphisms and clinical outcome in advanced gastric cancer patients treated with FOLFOX.

The insulin-like growth factor (IGF) axis plays a crucial role in proliferation, differentiation, migration, angiogenesis, and apoptosis. The present study evaluated the associations between IGF axis single-nucleotide polymorphisms (SNPs) and clinical outcomes in advanced gastric cancer (AGC) patients treated with oxaliplatin, 5-fluorouracil, and leucovorin (FOLFOX). A total of 190 patients undergoing FOLFOX chemotherapy for AGC were considered eligible for this study. Forty-four SNPs of 10 IGF axis genes were genotyped. Levels of serum IGF1 were measured using enzyme-linked immunoassays. SNPs of the IGF1R (rs12423791), and IGF1 (rs2162679, rs5742612, rs35767) genes were significantly associated with tumor response to FOLFOX. SNPs of rs4619 and rs17847203 were significantly associated with PFS (hazard ratio [HR] 0.575, 95% CI 0.385-0.858, P = 0.007; and HR 2.530, 95% CI 1.289-4.966, P = 0.007; respectively). SNPs of rs2872060 were significantly associated with OS-OS was shorter in patients carrying the TT variant than in those with the GG/GT genotypes (HR, 1.708, 95% CI 1.024-2.850, P = 0.040). The GT genotype of rs12847203 was also identified as an independent prognostic factor (HR 2.087, 95% CI 1.070-4.069, P = 0.031). These results suggest that IGF axis-pathway SNPs could be used as prognostic biomarkers of the outcome of FOLFOX chemotherapy in AGC patients. This information may facilitate identification of population subgroups that could benefit from IGF1R-targeted agents.

Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD.

Although autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease, and is characterized by the formation of multiple fluid-filled cysts, which results in renal failure, early diagnosis and treatment of ADPKD have yet to be defined. Herein, we observed that the promoter region of the gene encoding mucin-like protocadherin (MUPCDH) was hypermethylated in the renal tissue of patients with ADPKD compared to non-ADPKD controls. Inversely, MUPCDH was significantly repressed in ADPKD, especially in cyst-lining cells. Our results indicate that aberrant methylation of MUPCDH promoter CpG islands may be negatively correlated with reduced expression level of MUPCDH and that this contributes to abnormal cell proliferation in ADPKD. It suggests that methylation status of MUPCDH promoter can be used as a novel epigenetic biomarker and a therapeutic target in ADPKD.

SLC15A2 genomic variation is associated with the extraordinary response of sorafenib treatment: whole-genome analysis in patients with hepatocellular carcinoma.

Reliable biomarkers are required to predict the response to sorafenib. We investigated genomic variations associated with responsiveness to sorafenib for patients with unresectable hepatocellular carcinoma (HCC). Blood samples from 2 extreme, 2 strong and 3 poor responders to sorafenib were subjected to whole-genome analysis. Then, we validated candidate genomic variations with another 174 HCC patients, and performed in vitro functional analysis and in silico analyses. Genomic data of >96 gigabases/sample was generated at average of ~34X sequencing depth. In total, 1813 genomic variations were matched to sorafenib responses in clinical data; 708 were located within regions for sorafenib-target genes or drug absorption, distribution, metabolism, and excretion (ADME)-related genes. From them, 36 variants were within the coding regions and 6 identified as non-synonymous single-nucleotide variants from 4 ADME-related genes (ABCB1, FMO3, MUSK, and SLC15A2). Validation genotyping confirmed sequencing results and revealed patients genotype for rs2257212 in SLC15A2 showed longer progression-free survival (HR = 2.18). In vitro study displayed different response to sorafenib depending on the genotype of SLC15A2. Structural prediction analysis revealed changes of the phosphorylation levels in protein, potentially affecting sorafenib-associated enzymatic activity. Our finding using extreme responder seems to generate robust biomarker to predict the response of sorafenib treatment for HCC.

Clinical likelihood of sporadic primary EGFR T790M mutation in EGFR-mutant lung cancer.

BACKGROUND: It has been reported that the presence of pretreatment EGFR T790M mutation may reduce the efficacy to EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutant lung cancer. However, clinicopathologic features related to the likelihood of T790M mutation before treatment remains unknown. PATIENTS AND METHODS: DNA from 124 pretreatment tissue samples from patients with advanced non-small-cell lung cancer carrying sensitive EGFR mutations was genotyped for EGFR T790M mutation with mass spectrometry. We compared the characteristics of 24 T790M patients and 100 patients with no or a low-level T790M mutation. RESULTS: There were no differences in age, sex, histology, or initial stage between T790M and non/low T790M groups. However, there were significantly more never-smokers in the T790M group (P = .017). Brain metastasis was also more common in the T790M group (P = .036). The response rates to platinum, taxane, gemcitabine, and pemetrexed did not differ between the 2 groups. In the T790M group, the response rates were not significantly different among the 4 cytotoxic drugs (P = .809). The median time to progression during EGFR-TKI therapy was shorter in the T790M group than in the non/low T790M group (4.1 vs. 11.5 months, respectively; P < .001). The median overall survival from the start of first-line treatment of advanced disease was similar in both groups (31.5 vs. 36.0 months, respectively; P = .310). CONCLUSION: The clinical features of EGFR T790M-mutant lung cancer were similar to those of sensitive EGFR-mutant lung cancer, except for the overrepresentation of never-smokers and brain metastasis.

HOXA9 inhibits migration of lung cancer cells and its hypermethylation is associated with recurrence in non-small cell lung cancer.

This study was aimed at understanding the clinicopathological significance of HOXA9 hypermethylation in non-small cell lung cancer (NSCLC). HOXA9 hypermethylation was characterized in six lung cancer cell lines, and its clinicopathological significance was analyzed using methylation-specific PCR in 271 formalin-fixed paraffin-embedded tissues and 27 fresh-frozen tumor and matched normal tissues from 298 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA9 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA9 into H23 lung cancer cells resulted in the inhibition of cell migration but not proliferation. Conversely, sequence-specific siRNA-mediated knockdown of HOXA9 enhanced cell migration. The mRNA levels of HOXA9 in 27 fresh-frozen tumor tissues were significantly lower than in matched normal tissues (P<0.0001; Wilcoxon signed-rank test). HOXA9 hypermethylation was found in 191 (70%) of 271 primary NSCLCs. HOXA9 hypermethylation was not associated with tumor size (P=0.12) and Ki-67 proliferation index (P=0.15). However, patients with HOXA9 hypermethylation had poor recurrence-free survival (hazard ratio=3.98, 95% confidence interval = 1.07-17.09, P=0.01) in never-smokers, after adjusting for age, sex, tumor size, adjuvant therapy, pathologic stage, and histology. In conclusion, the present study suggests that HOXA9 inhibits migration of lung cancer cells and its hypermethylation is an independent prognostic factor for recurrence-free survival in never-smokers with NSCLC.

Lack of effects of peroxisome proliferator-activated receptor gamma genetic polymorphisms on breast cancer risk: a case-control study and pooled analysis.

A growing body of evidence suggests that the peroxisome proliferator-activated receptor-gamma (PPARγ) gene may harbor targets for the chemoprevention of breast cancer. However, it is unclear whether polymorphisms in the PPARγ gene are associated with the susceptibility of breast cancer. We performed a candidate gene association study between PPARγ polymorphisms and breast cancer and a meta-analysis on the association of breast cancer with selected PPARγ variants. Six single nucleotide polymorphisms (SNPs) in the PPARγ gene were analyzed among 456 breast cancer patients and 461 controls from the National Cancer Center in Korea. Association between the polymorphisms and breast cancer risk were assessed using the Cochrane-Armitage test for trend and a multivariate logistic regression model. Two SNPs, rs3856806 and rs1801282, had been previously analyzed, thus enabling us to perform pooled analyses on their associations with breast cancer susceptibility. Our findings from the candidate gene association study showed no association between the PPARγ gene polymorphisms and breast cancer risk. A meta-analysis combining existing studies and our current study also refuted an association of the PPARγ gene with breast cancer. Our findings suggest that the PPARγ gene may not harbor variants that alter breast cancer susceptibility, although a moderate sample size might have precluded a decisive conclusion.

Comparison of targeted next-generation sequencing with conventional sequencing for predicting the responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy in never-smokers with lung adenocarcinoma.

PURPOSE: To investigate the clinical utility of targeted next-generation sequencing (NGS) for predicting the responsiveness to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy, we compared the efficacy with conventional sequencing in never-smokers with lung adenocarcinoma (NSLAs). PATIENTS AND METHODS: We obtained DNA from 48 NSLAs who received gefitinib or erlotinib for their recurrent disease after surgery. Sanger sequencing and peptide nucleic acid clamp polymerase chain reaction (PCR) were used to analyze EGFR, KRAS, BRAF, and PIK3CA mutations. We analyzed ALK, RET, and ROS1 rearrangements by fluorescent in situ hybridization or reverse transcriptase-PCR and quantitative real-time PCR. After molecular screening, Ion Torrent NGS was performed in 31 cases harboring only EGFR exon 19 deletions (19DEL), an L858R mutation, or none of the above mutations. RESULTS: The 31 samples were divided into four groups: (1) responders to EGFR-TKIs with only 19DEL or L858R (n=15); (2) primary resistance to EGFR-TKI with only 19DEL or L858R (n=4); (3) primary resistance to EGFR-TKI without any mutations (n=8); (4) responders to EGFR-TKI without any mutations (n=4). With NGS, all conventionally detected mutations were confirmed except for one L858R in group 2. Additional uncovered predictive mutations with NGS included one PIK3CA E542K in group 2, two KRAS (G12V and G12D), one PIK3CA E542K, one concomitant PIK3CA and EGFR L858R in group 3, and one EGFR 19DEL in group 4. CONCLUSIONS: Targeted NGS provided a more accurate and clinically useful molecular classification of NSLAs. It may improve the efficacy of EGFR-TKI therapy in lung cancer.

Clinical outcome according to the level of preexisting epidermal growth factor receptor T790M mutation in patients with lung cancer harboring sensitive epidermal growth factor receptor mutations.

BACKGROUND: Epidermal growth factor receptor (EGFR) T790M mutation drives acquired drug resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in patients with EGFR-mutant lung cancer. However, it was reported that this mutation may exist before drug exposure. The objective of the current study was to evaluate whether the clinical outcomes are affected by the percentage of preexisting T790M mutations within a tumor. METHODS: Pretreatment tissues were collected from 124 patients with advanced non-small cell lung cancer with sensitizing EGFR mutations that were detected by direct sequencing. Genotyping for EGFR T790M mutation was further performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Patients who were positive for the T790M mutation were divided to 2 subgroups according to T790M mutant signal frequency. RESULTS: The T790M mutation was found in 31 patients (25.0%). The T790M mutation frequency at which the risk of disease progression after therapy with EGFR-TKIs begins to increase was estimated to be 3.2%. The patients with T790M-positive tumors had a shorter time to disease progression after treatment with EGFR-TKIs (median, 6.3 months vs 11.5 months; P < .001) and overall survival (median, 16.1 months vs 26.5 months; P = .065) compared with those with T790M-negative tumors. Among the T790M-positive patients, the patients with high T790M frequency (9 patients) were found to have a shorter time to disease progression (median, 2.4 months vs 6.7 months; P = .009) and overall survival (median, 9.1 months vs 18.7 months; P = .018) compared with those with low T790M frequency (22 patients). CONCLUSIONS: A preexisting EGFR T790M mutation was noted in 25% of patients with EGFR-mutant lung cancer. Patients with a high T790M mutation frequency had worse clinical outcomes to EGFR-TKIs than patients with a low T790M mutation frequency.

Genomic profile analysis of diffuse-type gastric cancers.

BACKGROUND: Stomach cancer is the third deadliest among all cancers worldwide. Although incidence of the intestinal-type gastric cancer has decreased, the incidence of diffuse-type is still increasing and its progression is notoriously aggressive. There is insufficient information on genome variations of diffuse-type gastric cancer because its cells are usually mixed with normal cells, and this low cellularity has made it difficult to analyze the genome. RESULTS: We analyze whole genomes and corresponding exomes of diffuse-type gastric cancer, using matched tumor and normal samples from 14 diffuse-type and five intestinal-type gastric cancer patients. Somatic variations found in the diffuse-type gastric cancer are compared to those of the intestinal-type and to previously reported variants. We determine the average exonic somatic mutation rate of the two types. We find associated candidate driver genes, and identify seven novel somatic mutations in CDH1, which is a well-known gastric cancer-associated gene. Three-dimensional structure analysis of the mutated E-cadherin protein suggests that these new somatic mutations could cause significant functional perturbations of critical calcium-binding sites in the EC1-2 junction. Chromosomal instability analysis shows that the MDM2 gene is amplified. After thorough structural analysis, a novel fusion gene TSC2-RNF216 is identified, which may simultaneously disrupt tumor-suppressive pathways and activate tumorigenesis. CONCLUSIONS: We report the genomic profile of diffuse-type gastric cancers including new somatic variations, a novel fusion gene, and amplification and deletion of certain chromosomal regions that contain oncogenes and tumor suppressors.

HOXA11 hypermethylation is associated with progression of non-small cell lung cancer.

This study was aimed at understanding the functional significance of HOXA11 hypermethylation in non-small cell lung cancer (NSCLC). HOXA11 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using formalin-fixed paraffin-embedded tissues from 317 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA11 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA11 into H23 lung cancer cells resulted in the inhibition of cell migration and proliferation. HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs. HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively). HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival. Patients with pT2 and pT3 stages were 1.85 times (95% confidence interval [CI] = 1.04-3.29; P = 0.04) and 5.47 times (95% CI = 1.18-25.50; P = 0.01), respectively, more likely to show HOXA11 hypermethylation than those with pT1 stage, after adjusting for age, sex, and histology. In conclusion, the present study suggests that HOXA11 hypermethylation may contribute to the progression of NSCLC by promoting cell proliferation or migration.