Changes in SLIT2 expression are associated with the migration of human ovarian clear cell carcinoma cells.

Ovarian clear cell carcinoma (OCCC) is characterized by a poor survival of patients, which is mainly due to metastasis and treatment failure. Slit guidance ligand 2 (SLIT2), a secreted protein, has been reported to modulate the migration of neural cells and human cancer cells. However, the effect of changes in SLIT2 expression on the regulation of cell migration in OCCC remains unknown. The present study examined alterations in SLIT2 expression using OCCC cell models, including low- and high-mobility SKOV3 cells, as well as OCCC tissues. DNA methylation analysis suggested that promoter hypermethylation was responsible for the low expression levels of SLIT2 in OCCC cells. The demethylating agent 5-Aza-deoxycytosine was able to restore SLIT2 expression at both the mRNA and protein levels in high-mobility SKOV3 cells that harbored the relevant methylated promoter. Overexpression of SLIT2 inhibited the migration of high-mobility OCCC cells, as well as decreased the protein expression levels of ß-catenin, phosphorylated (p)AKT and snail family transcriptional repressor 1 (SNAI1). On the other hand, knockdown of SLIT2 increased the migration of low-mobility OCCC cells, and enhanced the protein expression levels of ß-catenin, pAKT and SNAI1. Overall, the results of the present study provided evidence that low expression levels of SLIT2 were associated with increased OCCC cell migration, and that SLIT2 may act as a suppressor gene of cancer cell migration.

The Alteration of CTNNBIP1 in Lung Cancer.

ß-catenin is a major component of the Wnt/ß-catenin signaling pathway, and is known to play a role in lung tumorigenesis. ß-catenin-interacting protein 1 (CTNNBIP1) is a known repressor of ß-catenin transactivation. However, little is known about the role of CTNNBIP1 in lung cancer. The aim of this study was to carry out a molecular analysis of CTNNBIP1 and its effect on ß-catenin signaling, using samples from lung cancer patients and various lung cancer cell lines. Our results indicate a significant inverse correlation between the CTNNBIP1 mRNA expression levels and the CTNNBIP1 promoter hypermethylation, which suggests that the promoter hypermethylation is responsible for the low levels of CTNNBIP1 present in many lung cancer patient samples. The ectopic expression of CTNNBIP1 is able to reduce the ß-catenin transactivation; this then brings about a decrease in the expression of ß-catenin-targeted genes, such as matrix metalloproteinase 7 (MMP7). Conversely, CTNNBIP1 knockdown is able to increase ß-catenin transactivation and the expression of MMP7. In agreement with these findings, a low level of CTNNBIP1 was found to be correlated with a high level of MMP7 when a publicly available microarray dataset for lung cancer was analyzed. Also, in agreement with the above, the ectopic expression of CTNNBIP1 inhibits the migration of lung cancer cells, whereas the CTNNBIP1 knockdown increases cancer cell migration. Our findings suggest that CTNNBIP1 is a suppressor of cancer migration, thus making it a potential prognostic predictor for lung cancer.

Growth-arrest-specific 7C protein inhibits tumor metastasis via the N-WASP/FAK/F-actin and hnRNP U/ß-TrCP/ß-catenin pathways in lung cancer.

Growth-arrest-specific 7 (GAS7) belongs to a group of adaptor proteins that coordinate the actin cytoskeleton. Among human GAS7 isoforms, only GAS7C possesses a Src homology 3 domain. We report here that GAS7C acts as a migration suppressor and can serve as a prognostic biomarker in lung cancer. GAS7C overexpression reduces lung cancer migration, whereas GAS7C knockdown enhances cancer cell migration. Importantly, ectopically overexpressed GAS7C binds tightly with N-WASP thus inactivates the fibronectin/integrin/FAK pathway, which in turn leads to the suppression of F-actin dynamics. In addition, overexpression of GAS7C sequesters hnRNP U and thus decreases the level of ß-catenin protein via the ß-TrCP ubiquitin-degradation pathway. The anti-metastatic effect of GAS7C overexpression was also confirmed using lung cancer xenografts. Our clinical data indicated that 23.6% (25/106) of lung cancer patients showed low expression of GAS7C mRNA which correlated with a poorer overall survival. In addition, low GAS7C mRNA expression was detected in 60.0% of metastatic lung cancer patients, indicating an association between low GAS7C expression and cancer progression. A significant inverse correlation between mRNA expression and promoter hypermethylation was also found, which suggests that the low level of GAS7C expression was partly due to promoter hypermethylation. Our results provide novel evidence that low GAS7C correlates with poor prognosis and promotes metastasis in lung cancer. Low GAS7C increases cancer cell motility by promoting N-WASP/FAK/F-actin cytoskeleton dynamics. It also enhances ß-catenin stability via hnRNP U/ß-TrCP complex formation. Therefore, GAS7C acts as a metastasis suppressor in lung cancer.

Deregulation of SLIT2-mediated Cdc42 activity is associated with esophageal cancer metastasis and poor prognosis.

INTRODUCTION: SLIT2, a secreted protein, has been found to inactivate Cdc42 GTPase to modulate neural cell migration. However, alteration of SLIT2-mediated Cdc42 in terms of migration regulation remains undefined in esophageal squamous cell carcinoma (ESCC). METHODS: We report here in ESCC cell, animal, and clinical models that SLIT2 acts as a migration suppressor and serves as a prognostic biomarker. RESULTS: The immunohistochemistry data indicated that 31.8% (49 of 154) of tumors from ESCC patients showed low expression of SLIT2 protein which correlated with poor overall survival and disease-free survival. DNA methylation analysis suggested that promoter hypermethylation is responsible for low expression of SLIT2 in ESCC. Knockdown of SLIT2 increased ESCC cell migration, while SLIT2 stable overexpression reduced cell migration. ESCC cells treated with conditioned media from cells overexpressing SLIT2 also suppressed cell migration. Importantly, silencing of SLIT2 decreased the complex formation, and thus induced Cdc42 activity and promoted membrane localization of focal adhesion kinase and Paxillin. Anti-metastatic effect of SLIT2 was confirmed in an experimental metastasis model of SLIT2 knockdown ESCC cells. CONCLUSION: Our results provide novel evidence that low expression of SLIT2 correlates with poor prognosis and promotes metastasis in ESCC, which may be regulated by the Cdc42-mediated pathways.

HBP1 promoter methylation augments the oncogenic ß-catenin to correlate with prognosis in NSCLC.

ß-catenin nuclear accumulation is frequently identified in human non-small cell lung cancer (NSCLC). The HMG-box transcription factor 1 (HBP1) is a known repressor of ß-catenin transactivation. However, the role of HBP1 in relation to ß-catenin nuclear accumulation has not been addressed in human cancer patients. In addition, the mechanism of HBP1 gene alteration in NSCLC remains unclear, although HBP1 mutation and gene deletion of HBP1 are reported in breast and colon cancers. Here, we demonstrate that HBP1 acts as a tumour suppressor and serves as a prognostic biomarker in NSCLC clinical and cell models. The immunohistochemistry data indicated that 30.5% (25/82) of tumours from NSCLC patients showed absence or low expression of HBP1 protein. A significant inverse correlation between mRNA/protein expression and promoter hypermethylation suggested that promoter hypermethylation is responsible for low expression of HBP1 in NSCLC patients. Reactivation of HBP1 expression by demethylation reagent or ectopic expression of HBP1 suppressed ß-catenin transactivation. Conversely, HBP1 knockdown increased ß-catenin transactivation. Importantly, preserved expression of HBP1 had a significantly protective effect on prognosis in patients with ß-catenin nuclear accumulation, suggesting that low expression of HBP1 in NSCLC patients with ß-catenin nuclear accumulation was one of the major determinants of prognosis. Our data from cellular and clinical models suggest that HBP1 is a suppressor of cancer progression, making it a potential prognostic predictor and therapeutic target to attenuate lung cancer progression.

High seroprevalence of human herpesvirus type 8 in patients with end-stage renal disease in Taiwan.

BACKGROUND: Human herpesvirus type 8 (HHV-8) is the aetiologic agent of Kaposi's sarcoma (KS). The incidence of KS in renal transplant patients is much higher than in healthy controls. The risk is even higher among recipients seropositive for HHV-8 before transplantation. Patients with end-stage renal disease (ESRD) are immunocompromised and are candidates for renal transplantation, but HHV-8 seroprevalence in ESRD patients has not been well documented. OBJECTIVES: This study aimed to evaluate HHV-8 seroprevalence in ESRD patients in a cohort in Taiwan. STUDY DESIGN: Blood samples collected from 149 ESRD patients and 149 age- and sex-matched healthy controls were analysed for HHV-8 antibody with immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) and for HHV-8 DNA with polymerase chain reaction. RESULTS: Seropositivity and titres for HHV-8 antibodies with IFA as well as seropositivity with ELISA were significantly greater in ESRD patients than in healthy controls (P=0.006, 0.001 and 0.003, respectively). Patients with a history of taking herbal medicine had significantly greater ELISA positivity than those without such a history (P=0.004). ELISA positives, particularly patients, had much higher IFA antibody titres than ELISA negatives (P<0.0001). Seropositivity in ESRD patients was not related to lymphopaenia, monocytosis, dialysis duration or a history of transfusion. Two diabetic ESRD patients were positive for HHV-8 DNA. CONCLUSIONS: ESRD patients had significantly greater HHV-8 seropositivity than healthy controls in Taiwan. This association seems to be related to the geographic location of the cohort and invites further studies for the early association of HHV-8 infection in ESRD patients and risk for KS.

SLIT2 attenuation during lung cancer progression deregulates beta-catenin and E-cadherin and associates with poor prognosis.

Chromosome 4p15.3 is frequently deleted in late-stage lung cancer. We investigated the significance of the SLIT2 gene located in this region to lung cancer progression. SLIT2 encodes an extracellular glycoprotein that can suppress breast cancer by regulating beta-catenin. In this study, we examined alterations in the structure or expression of SLIT2, its receptor ROBO1, and beta-catenin, along with the AKT/glycogen synthase kinase 3beta (GSK3beta)/beta-transducin repeat-containing protein (betaTrCP) pathway in lung cancer cell lines and patients. Low SLIT2 expression correlated with an upward trend of pathological stage and poorer survival in lung cancer patients. Importantly, SLIT2, betaTrCP, and beta-catenin expression levels predicted postoperative recurrence of lung cancer in patients. Stimulating SLIT2 expression by various methods increased the level of E-cadherin caused by attenuation of its transcriptional repressor SNAI1. Conversely, knocking down SLIT2 expression increased cell migration and reduced cell adhesion through coordinated deregulation of beta-catenin and E-cadherin/SNAI1 in the AKT/GSK3beta/betaTrCP pathway. Our findings indicate that SLIT2 suppresses lung cancer progression, defining it as a novel "theranostic" factor with potential as a therapeutic target and prognostic predictor in lung cancer. Cancer Res; 70(2); 543-51.

Distinct HIC1-SIRT1-p53 loop deregulation in lung squamous carcinoma and adenocarcinoma patients.

A HIC1-SIRT1-p53 circular loop in which hypermethylation in cancer 1 (HIC1) represses the transcription of SIRT1 that deacetylates and inactivates p53 thus leading to HIC1 inactivation has been identified in cell and animal models. However, the alteration and prognostic effects of HIC1-SIRT1-p53 circular loop have never been demonstrated in human cancer patients. We examine the HIC1-SIRT1-p53 alterations in 118 lung cancer patients to define their etiological roles in tumorigenesis. We found that patients with lung squamous cell carcinoma with low p53 acetylation and SIRT1 expression mostly showed low HIC1 expression, confirming deregulation of HIC1-SIRT1-p53 circular loop in the clinical model. Interestingly, the expression of deleted in breast cancer 1 (DBC1), which blocks the interaction between SIRT1 deacetylase and p53, led to acetylated p53 in patients with lung adenocarcinoma. However, epigenetic alteration of HIC1 promoter by posttranslational modifications of histones and promoter hypermethylation favoring the compacted chromatin production attenuated the transcriptional induction by acetylated p53. Importantly, lung cancer patients with altered HIC1-SIRT1-p53 circular regulation showed poor prognosis. Our data show the first valid clinical evidence of the deregulation of HIC1-SIRT1-p53 loop in lung tumorigenesis and prognosis. Distinct status of p53 acetylation/deacetylation and HIC1 alteration mechanism result from different SIRT1-DBC1 control and epigenetic alteration in lung squamous cell carcinoma and lung adenocarcinoma.

Lung cancer susceptibility and prognosis associated with polymorphisms in the nonhomologous end-joining pathway genes: a multiple genotype-phenotype study.

BACKGROUND: Nonsmall cell lung cancer (NSCLC) frequently exhibits genomic instability, such as high fractional allelic loss (FAL). Genomic instability may result from unrepaired or misrepaired double-strand breaks (DSBs). The authors of this report postulated that polymorphisms in genes of the nonhomologous end-joining (NHEJ) pathway, which is the major DSB repair pathway in mammalian cells, may modulate lung cancer susceptibility and prognosis. METHODS: Patients with NSCLC (n = 152) and a group of appropriate age-matched and sex-matched controls (n = 162) were subjected to genotype analysis of the NHEJ pathway genes x-ray repair complementing defective repair in Chinese hamster cells 6 (Ku70) (reference single nucleotide polymorphism number [rs] 2267437), x-ray repair complementing defective repair in Chinese hamster cells 5 (Ku80) (rs3835), x-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4) (rs1805377), and DNA ligase IV (LIG4) (rs1805388). The gene-gene interaction (joint effect), genotype-environmental (ie, smoking) correlation, and genotype-phenotype (ie, FAL) correlation were examined. The Kaplan-Meier method and log-rank tests were used to assess the prognostic effect. RESULTS: There was a significant association between the XRCC4 and LIG4 genotypes with NSCLC risk in an analysis of individual polymorphism associations, and the risk of NSCLC increased further in a combined analysis of multiple polymorphisms (adjusted odds ratio [OR], 8.74). The patients who had a homozygous variant guanine/guanine genotype of the XRCC4 gene had a poorer prognosis compared with other patients (P = .015). There was a significant difference between the patient smokers and controls for XRCC4 (adjusted OR, 2.67) and LIG4 (adjusted OR, 2.04). In addition, polymorphisms in XRCC4 and LIG4 were linked significantly with patients who had high FAL (adjusted OR, 2.03-3.84). CONCLUSIONS: To the authors' knowledge, this is the first nested case-control study to demonstrate a significant association between the polymorphisms of genes in the NHEJ pathway and lung cancer susceptibility and prognosis. The results may be useful for risk assessment and disease monitoring of patients with NSCLC.

Frequent down-regulation of hRAB37 in metastatic tumor by genetic and epigenetic mechanisms in lung cancer.

We looked for the involvement tumor suppressor gene (TSG) in lung cancer in 17q25 region by loss of heterozygosity analysis and 5'/3' RACE and identified a candidate gene named human RAB37 (hRAB37), which encodes a small GTPase. The Ras-GTPase superfamily functions as important regulators including membrane trafficking and cytoskeletal organization. Therefore, we further examined the mRNA expression and promoter/exon1 hypermethylation of hRAB37 gene in paired normal and tumor lung tissue from 71 non-small cell lung cancer (NSCLC) patients. Low hRAB37 mRNA expression occurred in 47.9% (34/71) of patient and promoter/exon1 hypermethylation of hRAB37 was found in 57.7% (41/71) of patients. Low mRNA expression of hRAB37 was significantly associated with their promoter/exon1 hypermethylation. Importantly, a reduction in hRAB37 mRNA expression and promoter/exon1 hypermethylation was found to be significantly associated with lung metastatic patients as compared to non-metastatic patients. 5-Aza-2-deoxycytidine treatment of a highly metastatic cell line showed demethylation and re-expression of the hRAB37 gene and coincided with reduced migration. Knockdown of hRAB37 in low metastasis cell line led to a significant increase in cell migration. Our findings demonstrated that hRAB37 small GTPase and acts as a metastasis-related TSG in lung cancer. Promoter/exon1 methylation is the predominant mechanism in down-regulation of the hRAB37, and can serve as a potential prediction biomarker of NSCLC progression.

Minimal deletion regions in lung squamous cell carcinoma: association with abnormality of the DNA double-strand break repair genes and their applications on gene identification and prognostic biomarkers.

BACKGROUND: Lung squamous cell carcinoma (SCC) cells frequently exhibit markers of chromosome instability such as high fractional allelic loss (FAL). We postulated that alterations in the p53 damage responsive gene and in the double-strand break (DSB) repair genes, BRCA1 and XRCC5, are involved in patients with high FAL. In addition, chromosomal deletion analysis enables the delineation of the likely locations of tumor suppressor genes (TSG) and could provide molecular markers for disease classification. PATIENTS AND METHODS: To define the minimal deletion regions (MDRs), we used 92 microsatellites spanning 29 regions identified in our previous genome-wide chromosomal deletion study in 36 lung SCC patients to verify the maximal contiguous deletion loci. RESULTS: Eight MDRs at 2q35, 3p14.1-3p14.3, 3p22.2-p23, 3p25.3-3p26.3, 5q35.1-q35.2, 9p23-p24.1, 13q14.11-q14.2, and 17p13.1-p13.2 were found in lung SCC. The candidate genes GAS7 and OVCA2 in the MDR17pA (17p13.1-p13.2) were further examined for mRNA expression. Low expression of the GAS7 gene in 57% of patients analyzed suggested its importance in lung SCC tumorigenesis. In addition, we found a panel of five microsatellites (D3S1766, D4S2397, D4S2361, D13S175, and D17S974), which can be used as prognostic biomarkers in lung SCC. Furthermore, alteration in more than two genes in DSB repair-related pathways was more apparent in high FAL patients. CONCLUSIONS: Our results provide biomarkers that may be used for monitoring tumor progression and for positional cloning of new TSGs. Importantly, our data show direct evidence that alterations in DSB repair-related pathways are involved in the genomic instability verified by intensive microsatellites of lung SCC.

Epigenetic inactivation of the chromosomal stability control genes BRCA1, BRCA2, and XRCC5 in non-small cell lung cancer.

PURPOSE: Lung cancer cells frequently exhibit marked chromosome instability. We postulated that alterations of the double-strand break repair genes (BRCA1, BRCA2, and XRCC5) might be involved in lung cancer. PATIENTS AND METHODS: We examined the loss of protein and mRNA expression and the 5'CpG hypermethylation and allelic imbalance of the BRCA1, BRCA2, and XRCC5 genes in 98 non-small cell lung cancer (NSCLC) samples. Anchorage-dependent growth after reexpression of these genes was examined in a lung cancer cell line that originally lacked BRCA1 and BRCA2 expression. RESULTS: The data indicated that low protein expression of BRCA1 and BRCA2 was frequent in lung adenocarcinomas (42-44%), whereas low XRCC5 protein expression was more prevalent among squamous cell carcinoma (32%). In addition, low BRCA1 expression was significantly associated with low RB expression, especially in lung adenocarcinoma. Concurrent alterations in XRCC5 and p53 were the most frequent profiles in smoking patients. Importantly, low mRNA and protein expressions of BRCA1, BRCA2, and XRCC5 were significantly associated with their promoter hypermethylation. 5-Aza-2'-deoxycytidine treatment of NSCLC cells showed demethylation and reexpression of the BRCA1 and BRCA2 genes and reduced anchorage-independent growth. CONCLUSIONS: Our retrospective study provides compelling evidence that low mRNA and protein expression in the BRCA1/BRCA2 and XRCC5 genes occur in lung adenocarcinoma and squamous cell carcinoma, respectively, and that promoter hypermethylation is the predominant mechanism in deregulation of these genes. Alteration of the double-strand break repair pathway, perhaps by interacting with p53 and RB deregulation, is important in the pathogenesis of a subset of NSCLC.

Genomewide loss of heterozygosity and its clinical associations in non small cell lung cancer.

We extensively allelotyped a panel of 71 microdissected primary surgically resected non small cell lung cancer (NSCLC) tumors to identify chromosomal regions that are likely to contain tumor suppressor genes (TSGs) or associated with clinicopathologic and prognostic effects. Loss of heterozygosity (LOH) was detected by genotyping of 177 microsatellite markers and correlation of LOH with clinicopathologic parameters and prognosis was analyzed. Twenty markers showed an LOH frequency greater than 48%, and 8 of them (2p23.3, 2p24.3, 2q35, 6p22.2, 7p14.3, 7p22.2, 17q24.3 and 21q22.3) were novel in NSCLC. The high LOH regions were confirmed by further aligning continuous LOH regions from another set of 24 NSCLC tissues and defining 7 minimal deletion regions ranging from 1.29 to 12.26 cM. The aberrations of 8 markers showed a significant correlation with alteration of p16 and Rb proteins, suggesting the gene(s) located in the chromosomal loss that may interact with p16/Rb pathway. In addition, markers specifically associated with smoking, histology types and tumor stages were identified and the linked candidate TSGs were suggested. For example, marker D1S1612 closely linked with Mig-6 gene was associated with smoking patients, squamous cell carcinoma patients and late-stage patients. Furthermore, 3 markers, D2S2968, D6S2439 and D7S1818, were significantly associated with poor prognosis of NSCLC patients using both univariate and multivariate Cox's regression analyses (p = 0.035, 0.022 and 0.006, respectively). These markers can potentially be used for early lung cancer detection, outcome measurement and the positional cloning of new TSGs whose loss of function contributes to NSCLC tumorigenesis.

5' cytosine-phospho-guanine island methylation is responsible for p14ARF inactivation and inversely correlates with p53 overexpression in resected non-small cell lung cancer.

PURPOSE AND EXPERIMENTAL DESIGN: The molecular mechanisms by which the p14ARF gene is altered in non-small cell lung cancer (NSCLC) are complex and unclear. Using genetic and epigenetic analyses, we examined various molecular alterations including the loss of protein and mRNA expression, and 5'CpG hypermethylation, allelic imbalance, and mutation of the p14ARF gene in a series of 102 NSCLC samples, in parallel with clinicopathological and prognostic analyses. To clarify the biological significance of p14ARF alterations, its relationship with p16INK4a and p53 alterations was also examined. RESULTS: We found that 34% of NSCLC patients had aberrant P14ARF protein expression, which was more frequent in adenocarcinomas (AD; 44%) than in squamous cell carcinomas (22%; P = 0.024). A high concordance was observed between alterations in protein and mRNA expression and 5'CpG hypermethylation (P

Inactivation of hMLH1 and hMSH2 by promoter methylation in primary non-small cell lung tumors and matched sputum samples.

We performed a genetic and epigenetic study of the hMLH1 and hMSH2 mismatch repair genes in resected primary tumors from 77 non-small cell lung cancer (NSCLC) patients. The molecular alterations examined included the loss of mRNA and protein expression as well as promoter methylation, and the allelic imbalance of the chromosomal regions that harbor the genes. We found that 78% and 26% of patients showed at least one type of molecular alteration within the hMLH1 and hMSH2 genes, respectively. Promoter methylation of the hMLH1 gene was present in 55.8% of tumors, and was significantly associated with the reduction in mRNA and protein expression (P = 0.001). A 72% concordance of aberrant methylation in sputum samples with matched resected tumors was found. In addition, a 93% consistency between the promoter methylation and the mRNA expression of the hMSH2 gene was found in 14 female NSCLC patients. However, no correlation was found between the expression of hMLH1 and hMSH2 proteins and the allelic imbalance of five microsatellite markers closely linked to the genes. Our results suggest that hMLH1 is the major altered mismatch repair gene involved in NSCLC tumorigenesis, and that promoter methylation is the predominant mechanism in hMLH1 and hMSH2 deregulation. In addition, promoter methylation of the hMLH1 gene may be identified in sputum samples to serve as a potential diagnostic marker of NSCLC.

Alterations of the p16(ink4a) gene in resected nonsmall cell lung tumors and exfoliated cells within sputum.

Recently, we reported that p16 protein expression was nondetectable in 49.5% of 107 resected nonsmall cell lung cancers (NSCLCs), suggesting that the p16(INK4a) gene is frequently inactivated in primary NSCLC. To identify the molecular basis for this p16 immunohistochemical negativity further, we performed a genetic and epigenetic study of p16(INK4a) status in a series of 115 NSCLC samples parallel to the clinicopathologic and prognostic analyses. Microdissected tumor DNA samples were screened for homozygous deletion using comparative multiplex-polymerase chain reaction (PCR), for intragenic mutation using direct sequencing and for loss of heterozygosity (LOH) using an intragenic microsatellite marker, D9S942. Of these samples, 67 were further analyzed by SmaI-based PCR methylation assay to evaluate aberrant methylation at the gene. To examine the correlation of aberrant methylation in tumor and sputum samples, sputum samples from 12 matched patients were assessed for this change. We found that methylation of the p16(INK4a) gene was present in 38 of the 67 (56.7%) tumors and was significantly associated with negative p16 protein expression (p = 0.029). A 92% (11/12) concordance of sputum samples with matched resected tumors was found. The survival rates among adenocarcinoma patients with p16(INK4a) methylation were lower, but at a level of borderline significance compared with those patients without methylation (p = 0.071). In addition, 29.4% of the informative cases were found to harbor LOH at D9S942. None of the 115 microdissected tumors exhibited homozygous deletion in the p16(INK4a) gene. Only 1 patient exhibited a complex mutation at the fourth ankyrin repeat consensus sequence and concordantly demonstrated p16 immunohistochemical negativity. Overall, 69% (79/115) of NSCLC tumors had at least 1 type of p16(INK4a) alteration. Our data provide compelling evidence that p16(INK4a) alterations are involved in NSCLC tumorigenesis and that promoter methylation is the predominant mechanism in p16(INK4a) deregulation.

Loss of heterozygosity at loci of candidate tumor suppressor genes in microdissected primary non-small cell lung cancer.

To investigate the etiological association of allelic loss at chromosomal regions containing tumor suppressor genes (TSGs) in non-small cell lung cancer (NSCLC) in Taiwan, we examined 48 microdissected NSCLC samples for loss of heterozygosity (LOH) at nine loci where TSGs are localized nearby. The associations of LOH at each locus with clinicoparameters and prognosis were also examined. The frequent LOH was observed using markers, D3S1285 near the FHIT gene (58.3%), D17S938 near the p53 gene (56.7%), D9S925 near the p16 gene (54.5%), and D13S153 near the RB gene (47.6%). The occurrence of LOH at each TSG locus was compared with the patients' clinicoparameters. The incidence of LOH at D17S938 (p53 gene) and D3S4545 (VHL gene) was significantly higher in squamous carcinoma tumors than in adenocarcinoma tumors (P = 0.003 and 0.024, respectively). LOH of these two loci also occurred frequently in tumors from smoker patients compared to that from nonsmoker patients (P = 0.013 and 0.025, respectively). LOH at D13S153 (RB gene) was also associated with smoking (P = 0.008). In addition, the prognostic analyses indicated that the patients with LOH at D18S535 (18q21, near the SMAD2/4 gene) had significantly longer post-operative survival time compared to those without LOH (P = 0.03). Our results suggested that LOH at FHIT, p53, and p16 genes may occur frequently in NSCLC patients in Taiwan. In addition, LOH at p53, RB, and VHL may associate with smoking or squamous carcinoma patients and LOH at SMAD2/4 may be correlated with better prognosis.