<title>Patients and methods</title> <title>Lung cancer patients</title> <p>We reviewed the NSCLC surgery database maintained by the one cardiothoracic surgeon (BMC) for the period of 1994–2000. Patients who had received induction chemotherapy or for whom sufficient tissue was not available, were excluded. The final cohort had 155 (64.9%) men and 84 women (35.1%) with a median age at diagnosis of 68 years (range, 41–87 years) and a median survival time of 36.9 months (range, 1–113 months). Data on survival was obtained from the Cancer Registry of NSW, by routine follow-up visits or contact with the patient's general practitioner. Overall survival was measured from the date of surgery to the date of death or the date of last follow-up, censored patients being those who were alive at the time of last follow-up.</p> <p>This study cohort consisted of 92 (38.7%) adenocarcinomas (ADC), 54 (22.7%) large cell carcinomas (LCC), and 92 (38.7%) squamous cell carcinomas (SCC). These tumours were classified according to the American Joint Committee on Cancer (AJCC) tumour-node metastasis classification (##REF##12450554##Grondin and Liptay, 2002##) and consisted of 153 (64.0%) stage I and 86 (36.0%) stage II tumours (Table 2). The study was approved by the Ethics Review Committee of the Royal Prince Alfred Hospital (approval no. X02-0216).</p> <title>DNA extraction and bisulphite treatment</title> <p>Hematoxylin and Eosin-stained sections from paraffin-embedded tissue blocks were reviewed by an anatomical pathologist (WAC) for tumour and matching normal tissue specimens. Six to twelve serial 4 <italic>μ</italic>m sections of each block were used for DNA extraction, depending on the size of the tissue. DNA extraction was carried out using the Puregen Genomic DNA purification kit (Gentra Systems, MN, USA). Sodium bisulphite conversion was performed as previously described (##REF##12095267##Millar et al, 2002##).</p> <title>Expression of DLEC1 in lung cancer cell lines</title> <p>Five lung cancer cell lines, A427, A549, NCI-H292, NCI-H1299 and NCI-H358, were used. Total RNA and DNA were extracted from cell pellets using RNeasy® Mini Kit and DNeasy® Tissue Kit (Qiagen GmbH Inc., Germany), respectively. Normal human adult lung RNA samples were purchased from Stratagene (Stratagene, CA, USA). One microgram of RNA from each sample was used in a reverse transcription reaction using GeneAmp RNA PCR kit (Applied Biosystems, CA, USA). Expression of <italic>DLEC1</italic> was assessed by RT–PCR (DLEC1-F: 5′-TTCCTCCCTCGCCTACTC-3′; DLEC1-R: 5′-AAACTCATCCAGCCGCTG-3′). The primer pair was designed across exons 1 and 2 of the main <italic>DLEC1</italic> transcript NM_005106. <italic>GAPDH</italic> was used as control.</p> <p>To investigate if methylation regulates expression of <italic>DLEC1</italic>, cancer cells were treated with 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor. Freshly seeded cells were grown overnight in normal medium, which was then replaced with medium containing 1 <italic>μ</italic><sc>M</sc> of 5-aza (Sigma-Aldrich Corporation, MO, USA). Cells were allowed to grow for 72 h, with 5-aza-containing medium changed every 24 h, and harvested for DNA and RNA extraction. A cell viability of &gt;70% was retained after 72 h of treatment.</p> <title>Methylation-specific PCR</title> <p><italic>Deleted in lung and oesophageal cancer 1</italic> methylation status was assessed by a fluorescence based real-time detection quantitative methylation-specific PCR (MSP) with primers DLEC-m1, DLEC-m2 (##TAB##0##Table 1##) and a TaqMan® probe 5′-6FAM-TAATCAAACTTACGCTCACTTCGTCGCCG-BHQ1-3′ (Biosearch Technology, CA, USA) (##REF##16804544##Weisenberger et al, 2006##). A reference gene <italic>MYOD1</italic> was employed to normalise the DNA input of each sample as previously described (##REF##10344733##Eads et al, 1999##; ##REF##17260021##Kohonen-Corish et al, 2007##). Quantitative real-time PCR was performed for <italic>DLEC1</italic> and <italic>MYOD1</italic> in parallel using the RealMasterMix Probe ROX (Eppendorf, Hamburg, Germany) in the ABI7900HT Sequence Detection System (Applied Biosystems, CA, USA). <italic>Deleted in lung and oesophageal cancer 1</italic> methylation was scored as present when the value of (<italic>DLEC1</italic>/<italic>MYOD1</italic> × 100%)⩾5 or absent if the value is &lt;5. All samples were run in duplicate.</p> <p>Methylation-specific PCR of other chromosome 3p genes <italic>RARβ</italic>, <italic>MLH1</italic>, <italic>RASSF1A</italic> and <italic>BLU</italic> was carried out (##TAB##0##Table 1##) together with <italic>MYOD1</italic> amplification, as previously described (##REF##10344733##Eads et al, 1999##; ##REF##17260021##Kohonen-Corish et al, 2007##). PCR steps included 30 s for denaturing, annealing and extension (40 cycles), initial denaturation and final elongation for 10 min, and annealing temperatures of 55°C (<italic>MLH1</italic>), 57°C (<italic>MYOD1</italic>), 63°C (<italic>BLU</italic>), and 58°C (<italic>RARβ</italic>, <italic>RASSF1A</italic>).</p> <title>Immunohistochemistry and MSI analysis</title> <p>MLH1 expression on tissue microarrays was analysed as part of a previous study (##REF##18370958##Cooper et al, 2008##). Matched normal bronchial mucosa or peripheral lung parenchyma specimens were used as control tissue for each patient. MLH1 expression was scored semiquantitatively by multiplying the percentage of cells showing nuclear expression and the intensity of staining using a 3-tier grading system (1=weak, 2=moderate and 3=strong staining). Reduced MLH1 expression was taken for a score less than 100, of the maximum score of 300. MSI was analysed as previously described (##REF##15800322##Kohonen-Corish et al, 2005##, ##UREF##0##2006##), except that only two markers BAT25 and BAT26 were evaluated, which are sufficient for detecting high MSI (##REF##12454837##Suraweera et al, 2002##).</p> <title>Statistical and survival analysis</title> <p>Correlation between <italic>DLEC1</italic> methylation and clinicopathologic parameters was determined using the <italic>χ</italic><sup>2</sup> test while survival analysis was performed using the Kaplan–Meier log-rank and Cox Proportional Hazards Model in the StatView package, and <italic>P</italic>&lt;0.05 was regarded as statistically significant. Only those variables that were significant predictors of survival outcome in univariate analysis were incorporated into multivariate analyses.</p>
<title>Results</title> <title>High correlation between promoter methylation and loss of expression of DLEC1</title> <p>Expression of <italic>DLEC1</italic> was assessed by RT–PCR in five lung cancer cell lines. While <italic>DLEC1</italic> was expressed in normal lung tissue, no expression was detected in the A427, A549 and H1299 lung cancer cell lines (##FIG##0##Figure 1B##). We assessed <italic>DLEC1</italic> methylation using methylation-specific PCR (MSP). Only the methylated allele was detected in the three cell lines where <italic>DLEC1</italic> was not expressed, while both the unmethylated and methylated alleles were detected in cell lines expressing <italic>DLEC1</italic> (##FIG##0##Figure 1B##). Methylation was rare in normal lung tissue (2.5%, 200 specimens analysed). To determine whether methylation directly regulates the silencing of <italic>DLEC1</italic>, the cell line H1299 was treated with 5-aza, a DNA methyltransferase inhibitor. After 3 days of 5-aza treatment, <italic>DLEC1</italic> expression was restored and demethylation observed (##FIG##0##Figure 1C##).</p> <title>Promoter methylation of DLEC1, MLH1, RAR<italic>β</italic>, RASSF1A and BLU in lung cancer</title> <p>We employed MSP to assess the promoter methylation status of the five 3p candidate genes in 239 NSCLCs. Methylation was detected in 123 patients (51.5%) for <italic>RARβ</italic>, 86 (36.0%) for <italic>MLH1</italic>, 93 (38.9%) for <italic>DLEC1</italic>, 78 (32.6%) for <italic>RASSF1A</italic> and 85 (35.6%) for <italic>BLU</italic> (##TAB##1##Table 2##). Next we investigated the relationship between methylation of each set of two out of five genes. Significant correlation was observed between <italic>DLEC1</italic> and <italic>MLH1</italic> (<italic>P</italic>=0.0002), <italic>DLEC1</italic> and <italic>RASSF1A</italic> (<italic>P</italic>=0.0003), and <italic>RASSF1A</italic> and <italic>BLU</italic> methylation (<italic>P</italic>=0.017). <italic>MLH1</italic> methylation was also associated with <italic>RASSF1A</italic> (<italic>P</italic>=0.0006) and <italic>BLU</italic> (<italic>P</italic>=0.0005) (##TAB##2##Table 3##). Methylation of at least one of the five genes was detected in 204 of 239 (85.4%) patients; methylation of at least two genes in 139 (58.2%); three genes in 77 (32.2%); four genes in 36 (15.1%); and methylation of all five genes was detected in only nine (3.8%) patients.</p> <title>MLH1 expression in lung cancer tissue and MSI</title> <p>Expression of <italic>MLH1</italic> was previously determined using immunohistochemistry on tissue microarrays in 105 of the 239 patients (##REF##18370958##Cooper et al, 2008##). MSI was analysed in the whole cohort of 239 patients. Reduced <italic>MLH1</italic> expression was detected in seven of the 105 cancers including an apparent loss of MLH1 expression in two cancers, but none of the matching DNA specimens prepared from a larger area of the tumour showed any MSI using markers BAT25 and BAT26. Also, none of the seven cancers with reduced MLH1 expression showed <italic>MLH1</italic> promoter methylation. In the rest of the cohort MSI-H was detected in a stage 1B ADC (one marker) and a stage 2A LCC (both markers), of which only the latter was methylated in <italic>MLH1</italic>. There was no significant correlation between reduced MLH1 expression and survival (<italic>P</italic>=0.421).</p> <title>Methylation of DLEC1 and MLH1 are associated with poor patient survival</title> <p>A statistically significant association between methylation and histologic type was observed, where <italic>MLH1</italic> methylation had a higher frequency in SCC (45.6%) and LCC (40.7%) compared with ADC (22.8%); <italic>RASSF1A</italic> methylation was associated with LCC (53.7%); <italic>BLU</italic> and <italic>RARβ</italic> methylation with ADC (45.7% and 60.9%). Furthermore, <italic>MLH1</italic> and <italic>DLEC1</italic> methylation were associated with the presence of regional lymph-node metastases and AJCC stage II. No association was observed between methylation of the five genes and age of diagnosis, gender or tumour differentiation status, except that <italic>BLU</italic> methylation was more common in older patients (##TAB##1##Table 2##).</p> <p>Methylation of <italic>DLEC1</italic> (<italic>P</italic>=0.0005), <italic>MLH1</italic>, (<italic>P</italic>=0.004), and <italic>RASSF1A</italic> (<italic>P</italic>=0.024) as well as regional lymph node status (<italic>P</italic>&lt;0.0001) and AJCC stage (<italic>P</italic>&lt;0.0001) were associated with poorer overall survival (##FIG##1##Figure 2## and ##TAB##1##Table 2##). <italic>RARβ</italic> and <italic>BLU</italic> methylation were not prognostic in the whole NSCLC cohort using the Kaplan–Meier log-rank analysis (<italic>P</italic>=0.313 and 0.474). Regional lymph node metastases and AJCC stage are two of the known prognostic factors for NSCLC and these two parameters are dependent predictors of survival in our cohort. Therefore, a bivariate analysis with the molecular marker predictor (<italic>DLEC1</italic>, <italic>MLH1</italic> or <italic>RASSF1A</italic> methylation) and AJCC stage was set up. Methylation of either <italic>DLEC1</italic> or <italic>MLH1</italic> but not <italic>RASSF1A</italic> was a prognostic indicator independent of AJCC stage in the entire patient cohort (##TAB##3##Table 4##). <italic>Deleted in lung and oesophageal cancer 1</italic> methylation was also a prognostic factor independent of AJCC stage in the SCC subgroup of patients (HR, 1.754; 95% CI, 1.023–3.007; <italic>P</italic>=0.041) and <italic>MLH1</italic> methylation in LCC (HR, 2.926; 95% CI, 1.358–6.308; <italic>P</italic>=0.006).</p> <p>We then investigated if concordant methylation of two genes affect patient prognosis (##FIG##1##Figure 2##; ##TAB##3##Table 4##). Concordant <italic>MLH1</italic>/<italic>DLEC1</italic> methylation was associated with poorer overall survival in both univariate (HR, 2.075; 95% CI, 1.428–3.015; <italic>P</italic>=0.0001) and bivariate (HR, 1.668; 95% CI, 1.138–2.447; <italic>P</italic>=0.009) analyses. Also, <italic>MLH1</italic> methylation was prognostic in combination with <italic>RASSF1A</italic> methylation independent of AJCC stage in all patients (HR, 1.688, 95% CI, 1.127–2.529; <italic>P</italic>=0.011) and particularly in the LCC cohort (HR, 3.223; 95% CI, 1.482–7.008; <italic>P</italic>=0.003).</p>
<title>Discussion</title> <p><italic>Deleted in lung and oesophageal cancer 1</italic> is a candidate tumour suppressor gene in multiple cancers. Although the function of <italic>DLEC1</italic> is unclear, it suppresses tumour growth or reduces invasiveness of cancer cells (##REF##10213508##Daigo et al, 1999##; ##REF##16756719##Kwong et al, 2006##, ##REF##17099870##2007##). In this study, we demonstrate for the first time that the <italic>DLEC1</italic> promoter is methylated in lung cancer. The demethylating agent 5-aza reversed loss of mRNA expression in lung cancer cell lines. Frequent <italic>DLEC1</italic> methylation (34.2%) was observed in NSCLC and was most common in SCC (47.8%). <italic>DLEC1</italic> methylation was cancer-specific, as it was only rarely detected in matching normal lung tissue, and was strongly associated with stage II tumours and the spread of cancer to regional lymph nodes (<italic>P</italic>&lt;0.0001). <italic>DLEC1</italic> methylation was also associated with shorter overall survival in the whole cohort and in the SCC group of patients, and this remained statistically significant upon bivariate analysis with AJCC stage (##TAB##3##Table 4##). As there is no antibody available for <italic>DLEC1</italic>, we could not determine what proportion of methylated tumours would show loss or reduced DLEC1 protein expression. However, it has been previously demonstrated that <italic>DLEC1</italic> RNA expression was lost in eight of 30 primary lung cancers and that this was not due to gene mutations (##REF##10213508##Daigo et al, 1999##).</p> <p>The <italic>MLH1</italic> gene is located within 1 Mb of <italic>DLEC1</italic> in a locus that shows 55% LOH in NSCLC (##REF##9650548##Benachenhou et al, 1998##). Therefore, there has been some interest in determining the biological significance of reduced <italic>MLH1</italic> gene expression and promoter methylation in lung cancer. As gene alterations can cause either increased sensitivity or resistance of tumours to chemotherapy treatment, we excluded those patients who had received induction chemotherapy prior to surgery to avoid a possible bias in the molecular analyses. <italic>MLH1</italic> methylation was found in 36% of the cancers but did not result in the loss of gene expression in the 105 cancers analysed with immunohistochemistry. Only 6.7% of the cancers showed reduced MLH1 expression with stringent criteria (&lt;100 of the maximum score of 300) and none of these specimens were methylated. We also found that MLH1 methylation was patchy and/or monoallelic in region C of the MLH1 promoter by using combined bisulphite-restriction analysis (COBRA, ##REF##17909015##Hitchins et al, 2007##) (data not shown). This is consistent with the finding that MSI is extremely rare in NSCLC.</p> <p>It is intriguing therefore, that <italic>MLH1</italic> methylation showed strong prognostic significance, which is reported here for the first time. It was a marker of poor survival in the whole cohort, and particularly in the LCC subgroup, with both univariate and bivariate analyses. This is in contrast to colorectal adenocarcinoma where <italic>MLH1</italic> methylation causes the MSI-H phenotype, which has improved prognosis. There was a high correlation between <italic>DLEC1</italic> and <italic>MLH1</italic> methylation (<italic>P</italic>=0.0002). As for <italic>DLEC1</italic>, <italic>MLH1</italic> methylation was associated with stage II tumours and spread to regional lymph nodes. Concordant methylation of <italic>MLH1</italic> and <italic>DLEC1</italic> was also a marker of poor prognosis independent of stage in the whole cohort (##TAB##3##Table 4##).</p> <p>The close correlation between <italic>MLH1</italic> and <italic>DLEC1</italic> methylation may be a consequence or a byproduct of a long-range epigenetic effect in this region of chromosome 3p. The first such chromosomal region reported was 2q14.2, which shows modification of chromatin structure such as histone H3-K9 methylation in colon cancer cells. This results in clusters of both methylated and unmethylated genes being coordinately suppressed (##REF##16642018##Frigola et al, 2006##). It has recently been shown that <italic>DLEC1</italic> and <italic>MLH1</italic> are also subject to long-range epigenetic regulation in colon cancer (##REF##17909015##Hitchins et al, 2007##). Multiple genes in this region can be simultaneously silenced through promoter hypermethylation and histone methylation in MSI-positive colorectal cancers. This effect appears to extend centromeric from the <italic>MLH1</italic> gene and does not always reach <italic>DLEC1</italic> in all specimens. In bladder cancer there is also evidence of such long-range epigenetic regulation around the <italic>DLEC1</italic> gene, but here the predominant mechanism is gene silencing through histone methylation rather than CpG methylation, and <italic>MLH1</italic> was not analysed (##REF##17099711##Stransky et al, 2006##). The two genes, which were analysed in both studies, <italic>DLEC1</italic> and its neighbour <italic>PLCD1</italic>, are silenced through DNA methylation and H3-K9 dimethylation in colorectal cancer whereas in bladder cancer they are silenced through histone H3-K9 trimethylation. This suggests that there are tissue-specific differences in this regulation. Therefore, if such long-range epigenetic regulation of chromosome 3p is also operating in lung cancer, it is possible that some genes in the region may be affected less than others. As a consequence the overall methylation in this region could serve as a marker of poorer prognosis but only some genes show complete loss of function.</p> <p>The other three genes analysed in this study <italic>RASSF1A</italic>, <italic>BLU</italic> and <italic>RARβ</italic> are known to be methylated in lung cancer and all have shown functional characteristics of tumour suppressor genes (##REF##10717330##Toulouse et al, 2000##; ##REF##12024041##Shivakumar et al, 2002##; ##REF##12629521##Agathanggelou et al, 2003##). <italic>RARβ</italic> is located 12 Mb telomeric from <italic>MLH1</italic>, and <italic>RASSF1A</italic> and <italic>BLU</italic> about 12 Mb centromeric from <italic>DLEC1</italic>. <italic>RASSF1</italic> methylation was also highly correlated with <italic>DLEC1</italic> (<italic>P</italic>=0.0003) and <italic>MLH1</italic> methylation (<italic>P</italic>=0.0006), whereas <italic>RARβ</italic> was methylated independent of the other genes. The correlation between <italic>RASSF1A</italic> and <italic>BLU</italic> methylation observed here (<italic>P</italic>=0.017) has also been described previously (##REF##12629521##Agathanggelou et al, 2003##). However, none of these three markers were as strongly prognostic as <italic>DLEC1</italic> and <italic>MLH1</italic> methylation in this cohort. In a previous study <italic>RASSF1A</italic> methylation correlated with poor survival (##REF##12114441##Tomizawa et al, 2002##), but this has not been confirmed in all cohorts (##REF##15266335##Toyooka et al, 2004##; ##REF##15700308##Choi et al, 2005##). Here, <italic>RASSF1A</italic> methylation was a prognostic marker in univariate analyses but not independent of stage, as was also observed previously (##REF##15700308##Choi et al, 2005##). It was interesting that concordant methylation of <italic>MLH1</italic> with <italic>RASSF1</italic> was an independent marker of poor prognosis. This suggests that a possible long-range epigenetic effect may extend centromeric from the <italic>DLEC1</italic> locus but not telomeric from the <italic>MLH1</italic> locus.</p> <p>Taken together, our study has described two new prognostic markers, methylation of <italic>DLEC1</italic> and <italic>MLH1</italic> on chromosome 3p. Methylation of these two genes is clearly associated with each other and with methylation of <italic>RASSF1</italic> and <italic>BLU</italic>, which are ∼12 Mb centromeric from <italic>DLEC1</italic>. <italic>MLH1</italic> methylation itself does not lead to gene silencing in lung cancer and the biological significance of <italic>DLEC1</italic> methylation also needs further study. In any case, concordant methylation of <italic>MLH1</italic> with <italic>DLEC1</italic> or <italic>RASSF1A</italic> is a valuable prognostic indicator in lung cancer. Future studies should reveal whether <italic>DLEC1</italic>, another gene or perhaps multiple genes in this region are functionally the most important in lung carcinogenesis.</p>
<p>The significance of chromosome 3p gene alterations in lung cancer is poorly understood. This study set out to investigate promoter methylation in the <italic>deleted in lung and oesophageal cancer 1</italic> (<italic>DLEC1</italic>), <italic>MLH1</italic> and other 3p genes in 239 non-small cell lung carcinomas (NSCLC). <italic>DLEC1</italic> was methylated in 38.7%, <italic>MLH1</italic> in 35.7%, <italic>RARβ</italic> in 51.7%, <italic>RASSF1A</italic> in 32.4% and <italic>BLU</italic> in 35.3% of tumours. Any two of the gene alterations were associated with each other except <italic>RARβ</italic>. <italic>DLEC1</italic> methylation was an independent marker of poor survival in the whole cohort (<italic>P</italic>=0.025) and in squamous cell carcinoma (<italic>P</italic>=0.041). <italic>MLH1</italic> methylation was also prognostic, particularly in large cell cancer (<italic>P</italic>=0.006). Concordant methylation of <italic>DLEC1</italic>/<italic>MLH1</italic> was the strongest independent indicator of poor prognosis in the whole cohort (<italic>P</italic>=0.009). However, microsatellite instability and loss of MLH1 expression was rare, suggesting that <italic>MLH1</italic> promoter methylation does not usually lead to gene silencing in lung cancer. This is the first study describing the prognostic value of <italic>DLEC1</italic> and <italic>MLH1</italic> methylation in NSCLC. The concordant methylation is possibly a consequence of a long-range epigenetic effect in this region of chromosome 3p, which has recently been described in other cancers.</p>
<p>Lung cancer is one of the most common causes of cancer death. The overall 5-year survival rate for surgical resection of stage I non-small cell lung cancer (NSCLC) can achieve 60–75% while survival rates in stage II–IV patients remain poor. Unfortunately only a small subset responds to currently available treatments. Thus, it is important to identify and characterise new molecular markers and gene targets to improve the accuracy of prognosis and develop more targeted treatment strategies to improve the clinical management of lung cancer.</p> <p>Allelic loss of chromosome 3p is one of the most frequent and earliest documented events in lung cancer, with deletions at 3p24–26, 3p21.3, 3p21.1–21.2, 3p14.2 and 3p12–13, suggesting the presence of multiple tumour suppressor genes on 3p (##REF##7837389##Hung et al, 1995##; ##REF##10766185##Wistuba et al, 2000##; ##REF##12362274##Zabarovsky et al, 2002##). Recent work has revealed the involvement of frequent epigenetic alterations in the inactivation of many 3p candidate genes, including <italic>BLU</italic>, <italic>FHIT, RASSF1A</italic>, <italic>RARβ</italic> and <italic>SEMA3B</italic> (##REF##10888881##Dammann et al, 2000##; ##REF##10944551##Virmani et al, 2000##; ##REF##11325823##Zochbauer-Muller et al, 2001##; ##REF##12362274##Zabarovsky et al, 2002##; ##REF##15922865##Ito et al, 2005##). Detection of methylated genes in serum and sputum DNA from lung cancer patients has also raised the possibility of using DNA methylation as an early detection marker (##REF##9892187##Esteller et al, 1999##; ##REF##11085511##Palmisano et al, 2000##; ##REF##11956099##Belinsky et al, 2002##; ##REF##11809682##Usadel et al, 2002##).</p> <p>Methylation of the <italic>MLH1</italic> gene in 3p22.3 and its correlation with a mismatch repair defect and high microsatellite instability (MSI-H) is well characterised in sporadic colorectal cancer, where this phenotype is associated with better patient survival (##REF##16952542##Sinicrope et al, 2006##). In NSCLC <italic>MLH1</italic> methylation has been described with frequencies ranging from 7 to 59% (##REF##12841866##Yanagawa et al, 2003##; ##REF##15958624##Safar et al, 2005##) but in the absence of MSI-H (##REF##9650548##Benachenhou et al, 1998##; ##REF##15583832##Okuda et al, 2005##). LOH within the <italic>MLH1</italic> gene has also been detected in 55% (##REF##9650548##Benachenhou et al, 1998##) and reduced MLH1 expression in 59% of lung cancers (##REF##10945633##Xinarianos et al, 2000##). These intriguing findings have been followed by a recent report that <italic>MSH2</italic>, but not <italic>MLH1</italic>, methylation is a marker of poor prognosis in a Taiwanese cohort of nonsmoking female NSCLC patients (##REF##16061855##Hsu et al, 2005##). It remains to be determined if a mismatch repair gene defect has a role in lung carcinogenesis and why it is not associated with typical MSI-H.</p> <p>The <italic>deleted in lung and oesophageal cancer 1</italic> (<italic>DLEC1</italic>) gene is located about 1 Mb centromeric from <italic>MLH1</italic> (##FIG##0##Figure 1A##). The 3p21.3 region was identified as one of the common deleted regions in lung cancer. Four candidate genes in this region were analysed but no evidence of their involvement in cancer development was found (##REF##9179494##Ishikawa et al, 1997##). Further analysis led to the identification of the <italic>DLC1</italic> gene (##REF##10213508##Daigo et al, 1999##), which was later renamed <italic>DLEC1</italic>. Loss of <italic>DLEC1</italic> expression has been observed in lung, oesophageal, renal, ovarian and nasopharyngeal carcinoma cell lines and primary tumours and functional analyses strongly suggest that <italic>DLEC1</italic> is a tumour suppressor gene (##REF##10213508##Daigo et al, 1999##; ##REF##16756719##Kwong et al, 2006##, ##REF##17099870##2007##). Promoter hypermethylation has been shown to be responsible for silencing of <italic>DLEC1</italic> in ovarian cancer and in nasopharyngeal carcinoma (##REF##16756719##Kwong et al, 2006##, ##REF##17099870##2007##) but there has been no comprehensive methylation analysis reported for lung cancer.</p> <p>In this study, we investigated if promoter hypermethylation of <italic>DLEC1</italic> is found in lung cancer and whether it has any prognostic significance. We determined the relationship of <italic>DLEC1</italic> methylation with patient clinicopathologic variables and other 3p molecular markers, in particular <italic>MLH1, RARβ</italic>, <italic>RASSF1</italic> and <italic>BLU</italic> methylation.</p>
<p>We thank the National Health and Medical Research Council and the Cancer Institute NSW for funding, and the Australian Cancer Research Foundation for their sponsorship of the ACRF Unit for the Molecular Genetics of Cancer at the Garvan Institute. MKC is a Cancer Institute NSW Career Development Fellow and RLS an NHMRC Senior Principal Research Fellow.</p>
<fig id="fig1"><label>Figure 1</label><caption><p>(<bold>A</bold>) Schematic drawing of the short arm of chromosome 3 and the relative location of the <italic>RARβ</italic>, <italic>MLH1</italic>, <italic>DLEC1</italic>, <italic>RASSF1A</italic> and <italic>BLU</italic> genes. (<bold>B</bold>) <italic>DLEC1</italic> (NM_005106) and <italic>GAPDH</italic> expression using RT–PCR (two upper panels) and methylation status using MSP (two bottom panels) in lung cancer cell lines and in normal human lung tissue. (<bold>C</bold>) Restoration of <italic>DLEC1</italic> expression and concomitant demethylation of the CpG island in H1299 cells using the 5-aza treatment.</p></caption></fig> <fig id="fig2"><label>Figure 2</label><caption><p>Kaplan–Meier log-rank analysis of overall survival of NSCLC patients stratified by promoter methylation of (<bold>A</bold>) <italic>DLEC1</italic>; (<bold>B</bold>) <italic>MLH1</italic>, (<bold>C</bold>) <italic>RASSF1A</italic>, (<bold>D</bold>) <italic>RARβ</italic>, (<bold>E</bold>) <italic>BLU</italic>, (<bold>F</bold>) <italic>DLEC1/MLH1</italic>, (<bold>G</bold>) <italic>MLH1/RASSF1A</italic> and (<bold>H</bold>) <italic>DLEC1/RASSF1A</italic> (<sup>*</sup><italic>P</italic>&lt;0.05; <sup>**</sup><italic>P</italic>&lt;0.005; <sup>***</sup><italic>P</italic>&lt;0.0001).</p></caption></fig>
<table-wrap id="tbl1"><label>Table 1</label><caption><title>PCR primers used in the promoter methylation analysis of <italic>RARβ</italic>, <italic>MLH1</italic>, <italic>DLEC1</italic>, <italic>RASSF1A</italic> and <italic>BLU</italic></title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="left"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50">
<bold>Gene</bold>
</th><th align="left" valign="top" charoff="50">
<bold>Forward primer</bold>
</th><th align="left" valign="top" charoff="50">
<bold>Reverse primer</bold>
</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">
<italic>RARβ-m</italic>
<xref ref-type="fn" rid="t1-fn1">a</xref>
</td><td align="left" valign="top" charoff="50">5′-TCGAGAACGCGAGCGATTC-3′</td><td align="left" valign="top" charoff="50">5′-GACCAATCCAACCGAAACGA-3′</td></tr><tr><td align="left" valign="top" charoff="50">
<italic>MLH1-m</italic>
</td><td align="left" valign="top" charoff="50">5′-AGCGATTTTTAACGCGTAAGC-3′</td><td align="left" valign="top" charoff="50">5′-CTCAATACCTCGTACTCACG-3′</td></tr><tr><td align="left" valign="top" charoff="50">
<italic>DLEC1-m</italic>
</td><td align="left" valign="top" charoff="50">5′-TTTCGTTGCGTATTTAAGATATTTC-3′</td><td align="left" valign="top" charoff="50">5′-CGTAACGCTCATTCTCGCTACC-3′</td></tr><tr><td align="left" valign="top" charoff="50">
<italic>DLEC1-u</italic>
<xref ref-type="fn" rid="t1-fn2">b</xref>
</td><td align="left" valign="top" charoff="50">5′-TAGTTTTGTAGTTTGGTTTTGTT-3′</td><td align="left" valign="top" charoff="50">5′-ACAAAATATCTTAAATACACAACA-3′</td></tr><tr><td align="left" valign="top" charoff="50">
<italic>RASSF1A-m</italic>
</td><td align="left" valign="top" charoff="50">5′-TTAGCGAAGTACGGGTTTAATC-3′</td><td align="left" valign="top" charoff="50">5′-CTACCGTATAAAATTACACGCG-3′</td></tr><tr><td align="left" valign="top" charoff="50">
<italic>BLU-m</italic>
</td><td align="left" valign="top" charoff="50">5′-CGTGGGTTATAGTTCGAGAAAGC-3′</td><td align="left" valign="top" charoff="50">5′-AACGAATTAACCGCGCCTACGC-3′</td></tr><tr><td align="left" valign="top" charoff="50">
<italic>MYOD1</italic>
<xref ref-type="fn" rid="t1-fn3">c</xref>
</td><td align="left" valign="top" charoff="50">5′-CCAACTCCAAATCCCCTCTCTAT-3′</td><td align="left" valign="top" charoff="50">5′-TGATTAATTTAGATTGGGTTTAGAGAAGGA-3′</td></tr></tbody></table></table-wrap> <table-wrap id="tbl2"><label>Table 2</label><caption><title>Association of clinicopathologic variables with poor overall survival and with <italic>RARβ</italic>, <italic>MLH1</italic>, <italic>DLEC1</italic>, <italic>RASSF1A</italic> and <italic>BLU</italic> promoter methylation in the cohort of 239 NSCLC patients.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="char" valign="top" char="." charoff="50">
<bold>Kaplan– Meier log-rank</bold>
</th><th colspan="3" align="center" valign="top" char="." charoff="50">
<bold>RAR<italic>β</italic> methylation</bold>
</th><th colspan="3" align="center" valign="top" char="." charoff="50">
<bold>hMLH1 methylation</bold>
</th><th colspan="3" align="center" valign="top" char="." charoff="50">
<bold>DLEC1 methylation</bold>
</th><th colspan="3" align="center" valign="top" char="." charoff="50">
<bold>RASSF1A methylation</bold>
</th><th colspan="3" align="center" valign="top" char="." charoff="50">
<bold>BLU methylation</bold>
</th></tr><tr><th align="left" valign="top" charoff="50"> </th><th align="char" valign="top" char="." charoff="50"> </th><th colspan="3" align="center" valign="top" char="." charoff="50">
<bold>No. of patients</bold>
<hr/></th><th colspan="3" align="center" valign="top" char="." charoff="50">
<bold>No. of patients</bold>
<hr/></th><th colspan="3" align="center" valign="top" char="." charoff="50">
<bold>No. of patients</bold>
<hr/></th><th colspan="3" align="center" valign="top" char="." charoff="50">
<bold>No. of patients</bold>
<hr/></th><th colspan="3" align="center" valign="top" char="." charoff="50">
<bold>No. of patients</bold>
<hr/></th></tr><tr><th align="left" valign="top" charoff="50">
<bold>Clinicopathologic parameters</bold>
</th><th align="char" valign="top" char="." charoff="50">
<bold><italic>P</italic>-value</bold>
</th><th align="char" valign="top" char="." charoff="50">
<bold>+<xref ref-type="fn" rid="t2-fn1">a</xref></bold>
</th><th align="char" valign="top" char="." charoff="50">
<bold>−<xref ref-type="fn" rid="t2-fn2">b</xref></bold>
</th><th align="char" valign="top" char="." charoff="50">
<bold><italic>P</italic>-value</bold>
</th><th align="char" valign="top" char="." charoff="50">+</th><th align="char" valign="top" char="." charoff="50">−</th><th align="char" valign="top" char="." charoff="50">
<bold><italic>P</italic>-value</bold>
</th><th align="char" valign="top" char="." charoff="50">+</th><th align="char" valign="top" char="." charoff="50">−</th><th align="char" valign="top" char="." charoff="50">
<bold><italic>P</italic>-value</bold>
</th><th align="char" valign="top" char="." charoff="50">+</th><th align="char" valign="top" char="." charoff="50">−</th><th align="char" valign="top" char="." charoff="50">
<bold><italic>P</italic>-value</bold>
</th><th align="char" valign="top" char="." charoff="50">+</th><th align="char" valign="top" char="." charoff="50">−</th><th align="char" valign="top" char="." charoff="50">
<bold><italic>P</italic>-value</bold>
</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">
<italic>Age</italic>
</td><td align="char" valign="top" char="." charoff="50">0.5754</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">&lt;68 (<italic>n</italic>=117)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">63</td><td align="char" valign="top" char="." charoff="50">54</td><td align="char" valign="top" char="." charoff="50">0.4706</td><td align="char" valign="top" char="." charoff="50">39</td><td align="char" valign="top" char="." charoff="50">78</td><td align="char" valign="top" char="." charoff="50">0.4032</td><td align="char" valign="top" char="." charoff="50">49</td><td align="char" valign="top" char="." charoff="50">68</td><td align="char" valign="top" char="." charoff="50">0.3567</td><td align="char" valign="top" char="." charoff="50">41</td><td align="char" valign="top" char="." charoff="50">76</td><td align="char" valign="top" char="." charoff="50">0.4371</td><td align="char" valign="top" char="." charoff="50">34</td><td align="char" valign="top" char="." charoff="50">83</td><td align="char" valign="top" char="." charoff="50">0.0397</td></tr><tr><td align="left" valign="top" charoff="50">&gt;68 (<italic>n</italic>=122)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">60</td><td align="char" valign="top" char="." charoff="50">62</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">47</td><td align="char" valign="top" char="." charoff="50">75</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">44</td><td align="char" valign="top" char="." charoff="50">78</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">37</td><td align="char" valign="top" char="." charoff="50">85</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">51</td><td align="char" valign="top" char="." charoff="50">71</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">
<italic>Gender</italic>
</td><td align="char" valign="top" char="." charoff="50">0.1680</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Male (<italic>n</italic>=155)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">76</td><td align="char" valign="top" char="." charoff="50">79</td><td align="char" valign="top" char="." charoff="50">0.3068</td><td align="char" valign="top" char="." charoff="50">57</td><td align="char" valign="top" char="." charoff="50">98</td><td align="char" valign="top" char="." charoff="50">0.7293</td><td align="char" valign="top" char="." charoff="50">61</td><td align="char" valign="top" char="." charoff="50">94</td><td align="char" valign="top" char="." charoff="50">0.8488</td><td align="char" valign="top" char="." charoff="50">49</td><td align="char" valign="top" char="." charoff="50">106</td><td align="char" valign="top" char="." charoff="50">0.6468</td><td align="char" valign="top" char="." charoff="50">53</td><td align="char" valign="top" char="." charoff="50">102</td><td align="char" valign="top" char="." charoff="50">0.5475</td></tr><tr><td align="left" valign="top" charoff="50">Female (<italic>n</italic>=84)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">47</td><td align="char" valign="top" char="." charoff="50">37</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">29</td><td align="char" valign="top" char="." charoff="50">55</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">32</td><td align="char" valign="top" char="." charoff="50">52</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">29</td><td align="char" valign="top" char="." charoff="50">55</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">32</td><td align="char" valign="top" char="." charoff="50">52</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">
<italic>Tumour differentiation</italic>
</td><td align="char" valign="top" char="." charoff="50">0.2864</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Poor (<italic>n</italic>=103)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">48</td><td align="char" valign="top" char="." charoff="50">55</td><td align="char" valign="top" char="." charoff="50">0.3388</td><td align="char" valign="top" char="." charoff="50">44</td><td align="char" valign="top" char="." charoff="50">59</td><td align="char" valign="top" char="." charoff="50">0.3106</td><td align="char" valign="top" char="." charoff="50">44</td><td align="char" valign="top" char="." charoff="50">59</td><td align="char" valign="top" char="." charoff="50">0.7712</td><td align="char" valign="top" char="." charoff="50">35</td><td align="char" valign="top" char="." charoff="50">68</td><td align="char" valign="top" char="." charoff="50">0.3738</td><td align="char" valign="top" char="." charoff="50">34</td><td align="char" valign="top" char="." charoff="50">69</td><td align="char" valign="top" char="." charoff="50">0.2847</td></tr><tr><td align="left" valign="top" charoff="50">Moderate (<italic>n</italic>=113)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">65</td><td align="char" valign="top" char="." charoff="50">48</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">35</td><td align="char" valign="top" char="." charoff="50">78</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">41</td><td align="char" valign="top" char="." charoff="50">72</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">37</td><td align="char" valign="top" char="." charoff="50">76</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">41</td><td align="char" valign="top" char="." charoff="50">72</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Well (<italic>n</italic>=20)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">9</td><td align="char" valign="top" char="." charoff="50">11</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">6</td><td align="char" valign="top" char="." charoff="50">14</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">7</td><td align="char" valign="top" char="." charoff="50">13</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">4</td><td align="char" valign="top" char="." charoff="50">16</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">10</td><td align="char" valign="top" char="." charoff="50">10</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Unknown (<italic>n</italic>=3)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">1</td><td align="char" valign="top" char="." charoff="50">2</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">1</td><td align="char" valign="top" char="." charoff="50">2</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">1</td><td align="char" valign="top" char="." charoff="50">2</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">2</td><td align="char" valign="top" char="." charoff="50">1</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">0</td><td align="char" valign="top" char="." charoff="50">3</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">
<italic>Histologic type</italic>
</td><td align="char" valign="top" char="." charoff="50">0.5207</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">ADC (<italic>n</italic>=92)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">56</td><td align="char" valign="top" char="." charoff="50">36</td><td align="char" valign="top" char="." charoff="50">0.0317</td><td align="char" valign="top" char="." charoff="50">21</td><td align="char" valign="top" char="." charoff="50">71</td><td align="char" valign="top" char="." charoff="50">0.0047</td><td align="char" valign="top" char="." charoff="50">29</td><td align="char" valign="top" char="." charoff="50">63</td><td align="char" valign="top" char="." charoff="50">0.0696</td><td align="char" valign="top" char="." charoff="50">25</td><td align="char" valign="top" char="." charoff="50">67</td><td align="char" valign="top" char="." charoff="50">0.0008</td><td align="char" valign="top" char="." charoff="50">42</td><td align="char" valign="top" char="." charoff="50">50</td><td align="char" valign="top" char="." charoff="50">0.0278</td></tr><tr><td align="left" valign="top" charoff="50">LCC (<italic>n</italic>=54)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">20</td><td align="char" valign="top" char="." charoff="50">34</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">22</td><td align="char" valign="top" char="." charoff="50">32</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">19</td><td align="char" valign="top" char="." charoff="50">35</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">29</td><td align="char" valign="top" char="." charoff="50">25</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">17</td><td align="char" valign="top" char="." charoff="50">37</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">SCC (<italic>n</italic>=92)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">47</td><td align="char" valign="top" char="." charoff="50">45</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">42</td><td align="char" valign="top" char="." charoff="50">50</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">44</td><td align="char" valign="top" char="." charoff="50">48</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">23</td><td align="char" valign="top" char="." charoff="50">69</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">25</td><td align="char" valign="top" char="." charoff="50">67</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">
<italic>Primary tumour stage</italic>
</td><td align="char" valign="top" char="." charoff="50">0.1014</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">T1 (<italic>n</italic>=64)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">31</td><td align="char" valign="top" char="." charoff="50">33</td><td align="char" valign="top" char="." charoff="50">0.8517</td><td align="char" valign="top" char="." charoff="50">16</td><td align="char" valign="top" char="." charoff="50">48</td><td align="char" valign="top" char="." charoff="50">0.0995</td><td align="char" valign="top" char="." charoff="50">17</td><td align="char" valign="top" char="." charoff="50">47</td><td align="char" valign="top" char="." charoff="50">0.0555</td><td align="char" valign="top" char="." charoff="50">19</td><td align="char" valign="top" char="." charoff="50">45</td><td align="char" valign="top" char="." charoff="50">0.8406</td><td align="char" valign="top" char="." charoff="50">22</td><td align="char" valign="top" char="." charoff="50">42</td><td align="char" valign="top" char="." charoff="50">0.7303</td></tr><tr><td align="left" valign="top" charoff="50">T2 (<italic>n</italic>=154)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">81</td><td align="char" valign="top" char="." charoff="50">73</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">62</td><td align="char" valign="top" char="." charoff="50">92</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">66</td><td align="char" valign="top" char="." charoff="50">88</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">52</td><td align="char" valign="top" char="." charoff="50">102</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">57</td><td align="char" valign="top" char="." charoff="50">97</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">T3 (<italic>n</italic>=21)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">11</td><td align="char" valign="top" char="." charoff="50">10</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">8</td><td align="char" valign="top" char="." charoff="50">13</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">10</td><td align="char" valign="top" char="." charoff="50">11</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">7</td><td align="char" valign="top" char="." charoff="50">14</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">6</td><td align="char" valign="top" char="." charoff="50">15</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">
<italic>Regional lymph node status</italic>
</td><td align="char" valign="top" char="." charoff="50">&lt;0.0001</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">N0 (<italic>n</italic>=174)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">91</td><td align="char" valign="top" char="." charoff="50">83</td><td align="char" valign="top" char="." charoff="50">0.6728</td><td align="char" valign="top" char="." charoff="50">54</td><td align="char" valign="top" char="." charoff="50">120</td><td align="char" valign="top" char="." charoff="50">0.0091</td><td align="char" valign="top" char="." charoff="50">53</td><td align="char" valign="top" char="." charoff="50">121</td><td align="char" valign="top" char="." charoff="50">&lt;0.0001</td><td align="char" valign="top" char="." charoff="50">52</td><td align="char" valign="top" char="." charoff="50">122</td><td align="char" valign="top" char="." charoff="50">0.1378</td><td align="char" valign="top" char="." charoff="50">68</td><td align="char" valign="top" char="." charoff="50">106</td><td align="char" valign="top" char="." charoff="50">0.0632</td></tr><tr><td align="left" valign="top" charoff="50">N1 (<italic>n</italic>=65)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">32</td><td align="char" valign="top" char="." charoff="50">33</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">32</td><td align="char" valign="top" char="." charoff="50">33</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">40</td><td align="char" valign="top" char="." charoff="50">25</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">26</td><td align="char" valign="top" char="." charoff="50">39</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">17</td><td align="char" valign="top" char="." charoff="50">48</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">
<italic>AJCC staging</italic>
</td><td align="char" valign="top" char="." charoff="50">&lt;0.0001</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Stage I (<italic>n</italic>=153)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">80</td><td align="char" valign="top" char="." charoff="50">73</td><td align="char" valign="top" char="." charoff="50">0.7341</td><td align="char" valign="top" char="." charoff="50">46</td><td align="char" valign="top" char="." charoff="50">107</td><td align="char" valign="top" char="." charoff="50">0.0110</td><td align="char" valign="top" char="." charoff="50">43</td><td align="char" valign="top" char="." charoff="50">110</td><td align="char" valign="top" char="." charoff="50">&lt;0.0001</td><td align="char" valign="top" char="." charoff="50">45</td><td align="char" valign="top" char="." charoff="50">108</td><td align="char" valign="top" char="." charoff="50">0.1562</td><td align="char" valign="top" char="." charoff="50">62</td><td align="char" valign="top" char="." charoff="50">91</td><td align="char" valign="top" char="." charoff="50">0.0327</td></tr><tr><td align="left" valign="top" charoff="50">Stage II (<italic>n</italic>=86)</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">43</td><td align="char" valign="top" char="." charoff="50">43</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">40</td><td align="char" valign="top" char="." charoff="50">46</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">50</td><td align="char" valign="top" char="." charoff="50">36</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">33</td><td align="char" valign="top" char="." charoff="50">53</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">23</td><td align="char" valign="top" char="." charoff="50">63</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Total</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">123</td><td align="char" valign="top" char="." charoff="50">116</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">86</td><td align="char" valign="top" char="." charoff="50">153</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">93</td><td align="char" valign="top" char="." charoff="50">146</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">78</td><td align="char" valign="top" char="." charoff="50">161</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">85</td><td align="char" valign="top" char="." charoff="50">154</td><td align="char" valign="top" char="." charoff="50"> </td></tr></tbody></table></table-wrap> <table-wrap id="tbl3"><label>Table 3</label><caption><title><italic>P</italic>-values for pairwise correlation of promoter methylation in the 3p genes</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" char="." charoff="50">
<bold>
<italic>MLH1</italic>
</bold>
</th><th align="center" valign="top" char="." charoff="50">
<bold>
<italic>DLEC1</italic>
</bold>
</th><th align="center" valign="top" char="." charoff="50">
<bold>
<italic>RASSF1A</italic>
</bold>
</th><th align="center" valign="top" char="." charoff="50">
<bold>
<italic>BLU</italic>
</bold>
</th></tr><tr><th align="left" valign="top" charoff="50"> </th><th align="char" valign="top" char="." charoff="50">
<bold><italic>P</italic>-value</bold>
</th><th align="char" valign="top" char="." charoff="50">
<bold><italic>P</italic>-value</bold>
</th><th align="char" valign="top" char="." charoff="50">
<bold><italic>P</italic>-value</bold>
</th><th align="char" valign="top" char="." charoff="50">
<bold><italic>P</italic>-value</bold>
</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">
<italic>RARβ</italic>
</td><td align="char" valign="top" char="." charoff="50">0.0691</td><td align="char" valign="top" char="." charoff="50">0.1032</td><td align="char" valign="top" char="." charoff="50">0.5543</td><td align="char" valign="top" char="." charoff="50">0.7343</td></tr><tr><td align="left" valign="top" charoff="50">
<italic>MLH1</italic>
</td><td align="char" valign="top" char="." charoff="50">—</td><td align="char" valign="top" char="." charoff="50">0.0002</td><td align="char" valign="top" char="." charoff="50">0.0006</td><td align="char" valign="top" char="." charoff="50">0.0005</td></tr><tr><td align="left" valign="top" charoff="50">
<italic>DLEC1</italic>
</td><td align="char" valign="top" char="." charoff="50">—</td><td align="char" valign="top" char="." charoff="50">—</td><td align="char" valign="top" char="." charoff="50">0.0003</td><td align="char" valign="top" char="." charoff="50">0.0550</td></tr><tr><td align="left" valign="top" charoff="50">
<italic>RASSF1A</italic>
</td><td align="char" valign="top" char="." charoff="50">—</td><td align="char" valign="top" char="." charoff="50">—</td><td align="char" valign="top" char="." charoff="50">—</td><td align="char" valign="top" char="." charoff="50">0.0173</td></tr></tbody></table></table-wrap> <table-wrap id="tbl4"><label>Table 4</label><caption><title>Association of gene promoter methylation with survival in a univariate and a bivariate analysis, which takes into account AJCC stage of cancer</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/><col align="char" char="."/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th colspan="8" align="center" valign="top" char="." charoff="50">
<bold>Cox Proportional Regression Model</bold>
<hr/></th></tr><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" char="." charoff="50">
<bold>Univariate</bold>
</th><th align="center" valign="top" char="." charoff="50">
<bold>Bivariate</bold>
</th><th align="center" valign="top" char="." charoff="50">
<bold>Univariate</bold>
</th><th align="center" valign="top" char="." charoff="50">
<bold>Bivariate</bold>
</th><th align="center" valign="top" char="." charoff="50">
<bold>Univariate</bold>
</th><th align="center" valign="top" char="." charoff="50">
<bold>Bivariate</bold>
</th><th align="center" valign="top" char="." charoff="50">
<bold>Univariate</bold>
</th><th align="center" valign="top" char="." charoff="50">
<bold>Bivariate</bold>
</th></tr><tr><th align="left" valign="top" charoff="50">
<bold>Clinical or molecular marker</bold>
</th><th colspan="2" align="center" valign="top" char="." charoff="50">
<bold>All samples (No.=239)</bold>
</th><th colspan="2" align="center" valign="top" char="." charoff="50">
<bold>ADC (No.=92)</bold>
</th><th colspan="2" align="center" valign="top" char="." charoff="50">
<bold>LCC (No.=54)</bold>
</th><th colspan="2" align="center" valign="top" char="." charoff="50">
<bold>SCC (No.=92)</bold>
</th></tr></thead><tbody valign="top"><tr><td colspan="9" align="left" valign="top" charoff="50">
<italic>AJCC stage</italic>
</td></tr><tr><td align="left" valign="top" charoff="50"> HR</td><td align="char" valign="top" char="." charoff="50">2.795</td><td align="char" valign="top" char="." charoff="50">2.580–2.728</td><td align="char" valign="top" char="." charoff="50">3.126</td><td align="char" valign="top" char="." charoff="50">2.767–3.042</td><td align="char" valign="top" char="." charoff="50">2.930</td><td align="char" valign="top" char="." charoff="50">2.438–3.256</td><td align="char" valign="top" char="." charoff="50">2.594</td><td align="char" valign="top" char="." charoff="50">2.411–2.597</td></tr><tr><td align="left" valign="top" charoff="50"> 95% CI</td><td align="char" valign="top" char="." charoff="50">2.009–3.889</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">1.808–5.404</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">1.417–6.056</td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50">1.506–4.467</td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">
<italic> P</italic>
</td><td align="char" valign="top" char="." charoff="50">&lt;0.0001</td><td align="char" valign="top" char="." charoff="50">&lt;0.0001</td><td align="char" valign="top" char="." charoff="50">&lt;0.0001</td><td align="char" valign="top" char="." charoff="50">&lt;0.0007</td><td align="char" valign="top" char="." charoff="50">0.004</td><td align="char" valign="top" char="." charoff="50">&lt;0.021</td><td align="char" valign="top" char="." charoff="50">0.0006</td><td align="char" valign="top" char="." charoff="50">&lt;0.002</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td colspan="9" align="left" valign="top" charoff="50">
<italic>MLH1/DLEC1</italic>
</td></tr><tr><td align="left" valign="top" charoff="50"> HR</td><td align="char" valign="top" char="." charoff="50">2.075</td><td align="char" valign="top" char="." charoff="50">1.668</td><td align="char" valign="top" char="." charoff="50">2.347</td><td align="char" valign="top" char="." charoff="50">1.415</td><td align="char" valign="top" char="." charoff="50">2.146</td><td align="char" valign="top" char="." charoff="50">2.284</td><td align="char" valign="top" char="." charoff="50">1.731</td><td align="char" valign="top" char="." charoff="50">1.525</td></tr><tr><td align="left" valign="top" charoff="50"> 95% CI</td><td align="char" valign="top" char="." charoff="50">1.428–3.015</td><td align="char" valign="top" char="." charoff="50">1.138–2.447</td><td align="char" valign="top" char="." charoff="50">1.210–4.555</td><td align="char" valign="top" char="." charoff="50">0.683–2.932</td><td align="char" valign="top" char="." charoff="50">0.939–4.906</td><td align="char" valign="top" char="." charoff="50">0.992–5.257</td><td align="char" valign="top" char="." charoff="50">0.984–3.043</td><td align="char" valign="top" char="." charoff="50">0.863–2.696</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>P</italic></td><td align="char" valign="top" char="." charoff="50">0.0001</td><td align="char" valign="top" char="." charoff="50">0.009</td><td align="char" valign="top" char="." charoff="50">0.012</td><td align="char" valign="top" char="." charoff="50">0.350</td><td align="char" valign="top" char="." charoff="50">0.070</td><td align="char" valign="top" char="." charoff="50">0.052</td><td align="char" valign="top" char="." charoff="50">0.057</td><td align="char" valign="top" char="." charoff="50">0.146</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td colspan="9" align="left" valign="top" charoff="50">
<italic>MLH1/RASSF1A</italic>
</td></tr><tr><td align="left" valign="top" charoff="50"> HR</td><td align="char" valign="top" char="." charoff="50">1.951</td><td align="char" valign="top" char="." charoff="50">1.688</td><td align="char" valign="top" char="." charoff="50">1.702</td><td align="char" valign="top" char="." charoff="50">1.245</td><td align="char" valign="top" char="." charoff="50">3.011</td><td align="char" valign="top" char="." charoff="50">3.223</td><td align="char" valign="top" char="." charoff="50">1.390</td><td align="char" valign="top" char="." charoff="50">1.206</td></tr><tr><td align="left" valign="top" charoff="50"> 95% CI</td><td align="char" valign="top" char="." charoff="50">1.307–2.913</td><td align="char" valign="top" char="." charoff="50">1.127–2.529</td><td align="char" valign="top" char="." charoff="50">0.832–3.483</td><td align="char" valign="top" char="." charoff="50">0.595–2.607</td><td align="char" valign="top" char="." charoff="50">1.395–6.501</td><td align="char" valign="top" char="." charoff="50">1.482–7.008</td><td align="char" valign="top" char="." charoff="50">0.677–2.854</td><td align="char" valign="top" char="." charoff="50">0.586–2.482</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>P</italic></td><td align="char" valign="top" char="." charoff="50">0.001</td><td align="char" valign="top" char="." charoff="50">0.011</td><td align="char" valign="top" char="." charoff="50">0.146</td><td align="char" valign="top" char="." charoff="50">0.561</td><td align="char" valign="top" char="." charoff="50">0.005</td><td align="char" valign="top" char="." charoff="50">0.003</td><td align="char" valign="top" char="." charoff="50">0.370</td><td align="char" valign="top" char="." charoff="50">0.611</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td colspan="9" align="left" valign="top" charoff="50">
<italic>DLEC1/RASSF1A</italic>
</td></tr><tr><td align="left" valign="top" charoff="50"> HR</td><td align="char" valign="top" char="." charoff="50">1.505</td><td align="char" valign="top" char="." charoff="50">1.137</td><td align="char" valign="top" char="." charoff="50">1.763</td><td align="char" valign="top" char="." charoff="50">1.102</td><td align="char" valign="top" char="." charoff="50">1.292</td><td align="char" valign="top" char="." charoff="50">0.925</td><td align="char" valign="top" char="." charoff="50">1.338</td><td align="char" valign="top" char="." charoff="50">1.213</td></tr><tr><td align="left" valign="top" charoff="50"> 95% CI</td><td align="char" valign="top" char="." charoff="50">1.015–2.233</td><td align="char" valign="top" char="." charoff="50">0.759–1.705</td><td align="char" valign="top" char="." charoff="50">0.927–3.352</td><td align="char" valign="top" char="." charoff="50">0.548–2.214</td><td align="char" valign="top" char="." charoff="50">0.574–2.909</td><td align="char" valign="top" char="." charoff="50">0.395–2.163</td><td align="char" valign="top" char="." charoff="50">0.686–2.607</td><td align="char" valign="top" char="." charoff="50">0.623–2.362</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>P</italic></td><td align="char" valign="top" char="." charoff="50">0.042</td><td align="char" valign="top" char="." charoff="50">0.533</td><td align="char" valign="top" char="." charoff="50">0.084</td><td align="char" valign="top" char="." charoff="50">0.785</td><td align="char" valign="top" char="." charoff="50">0.536</td><td align="char" valign="top" char="." charoff="50">0.857</td><td align="char" valign="top" char="." charoff="50">0.393</td><td align="char" valign="top" char="." charoff="50">0.571</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td colspan="9" align="left" valign="top" charoff="50">
<italic>MLH1</italic>
</td></tr><tr><td align="left" valign="top" charoff="50"> HR</td><td align="char" valign="top" char="." charoff="50">1.621</td><td align="char" valign="top" char="." charoff="50">1.421</td><td align="char" valign="top" char="." charoff="50">2.034</td><td align="char" valign="top" char="." charoff="50">1.512</td><td align="char" valign="top" char="." charoff="50">2.591</td><td align="char" valign="top" char="." charoff="50">2.926</td><td align="char" valign="top" char="." charoff="50">1.007</td><td align="char" valign="top" char="." charoff="50">0.972</td></tr><tr><td align="left" valign="top" charoff="50"> 95% CI</td><td align="char" valign="top" char="." charoff="50">1.164–2.257</td><td align="char" valign="top" char="." charoff="50">1.016–1.988</td><td align="char" valign="top" char="." charoff="50">1.171–3.535</td><td align="char" valign="top" char="." charoff="50">0.842–2.714</td><td align="char" valign="top" char="." charoff="50">1.220–5.499</td><td align="char" valign="top" char="." charoff="50">1.358–6.308</td><td align="char" valign="top" char="." charoff="50">0.597–1.697</td><td align="char" valign="top" char="." charoff="50">0.576–1.640</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>P</italic></td><td align="char" valign="top" char="." charoff="50">0.004</td><td align="char" valign="top" char="." charoff="50">0.040</td><td align="char" valign="top" char="." charoff="50">0.012</td><td align="char" valign="top" char="." charoff="50">0.166</td><td align="char" valign="top" char="." charoff="50">0.013</td><td align="char" valign="top" char="." charoff="50">0.006</td><td align="char" valign="top" char="." charoff="50">0.980</td><td align="char" valign="top" char="." charoff="50">0.915</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td colspan="9" align="left" valign="top" charoff="50">
<italic>DLEC1</italic>
</td></tr><tr><td align="left" valign="top" charoff="50"> HR</td><td align="char" valign="top" char="." charoff="50">1.783</td><td align="char" valign="top" char="." charoff="50">1.471</td><td align="char" valign="top" char="." charoff="50">1.681</td><td align="char" valign="top" char="." charoff="50">1.337</td><td align="char" valign="top" char="." charoff="50">1.454</td><td align="char" valign="top" char="." charoff="50">1.103</td><td align="char" valign="top" char="." charoff="50">1.968</td><td align="char" valign="top" char="." charoff="50">1.754</td></tr><tr><td align="left" valign="top" charoff="50"> 95% CI</td><td align="char" valign="top" char="." charoff="50">1.283–2.479</td><td align="char" valign="top" char="." charoff="50">1.050–2.062</td><td align="char" valign="top" char="." charoff="50">0.983–2.872</td><td align="char" valign="top" char="." charoff="50">0.766–2.333</td><td align="char" valign="top" char="." charoff="50">0.698–3.029</td><td align="char" valign="top" char="." charoff="50">0.514–2.366</td><td align="char" valign="top" char="." charoff="50">1.155–3.353</td><td align="char" valign="top" char="." charoff="50">1.023–3.007</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>P</italic></td><td align="char" valign="top" char="." charoff="50">0.0006</td><td align="char" valign="top" char="." charoff="50">0.025</td><td align="char" valign="top" char="." charoff="50">0.058</td><td align="char" valign="top" char="." charoff="50">0.306</td><td align="char" valign="top" char="." charoff="50">0.317</td><td align="char" valign="top" char="." charoff="50">0.802</td><td align="char" valign="top" char="." charoff="50">0.013</td><td align="char" valign="top" char="." charoff="50">0.041</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td><td align="char" valign="top" char="." charoff="50"> </td></tr><tr><td colspan="9" align="left" valign="top" charoff="50">
<italic>RASSF1A</italic>
</td></tr><tr><td align="left" valign="top" charoff="50"> HR</td><td align="char" valign="top" char="." charoff="50">1.474</td><td align="char" valign="top" char="." charoff="50">1.259</td><td align="char" valign="top" char="." charoff="50">1.397</td><td align="char" valign="top" char="." charoff="50">1.174</td><td align="char" valign="top" char="." charoff="50">2.453</td><td align="char" valign="top" char="." charoff="50">1.902</td><td align="char" valign="top" char="." charoff="50">1.138</td><td align="char" valign="top" char="." charoff="50">1.021</td></tr><tr><td align="left" valign="top" charoff="50"> 95% CI</td><td align="char" valign="top" char="." charoff="50">1.049–2.071</td><td align="char" valign="top" char="." charoff="50">0.892–1.776</td><td align="char" valign="top" char="." charoff="50">0.796–2.451</td><td align="char" valign="top" char="." charoff="50">0.661–2.084</td><td align="char" valign="top" char="." charoff="50">1.133–5.310</td><td align="char" valign="top" char="." charoff="50">0.856–4.230</td><td align="char" valign="top" char="." charoff="50">0.620–2.089</td><td align="char" valign="top" char="." charoff="50">0.556–1.876</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>P</italic></td><td align="char" valign="top" char="." charoff="50">0.025</td><td align="char" valign="top" char="." charoff="50">0.190</td><td align="char" valign="top" char="." charoff="50">0.244</td><td align="char" valign="top" char="." charoff="50">0.584</td><td align="char" valign="top" char="." charoff="50">0.023</td><td align="char" valign="top" char="." charoff="50">0.115</td><td align="char" valign="top" char="." charoff="50">0.676</td><td align="char" valign="top" char="." charoff="50">0.947</td></tr></tbody></table></table-wrap>
<table-wrap-foot><fn id="t1-fn1"><label>a</label><p>Primers specific for methylated, bisulphite converted DNA.</p></fn><fn id="t1-fn2"><label>b</label><p>Primers specific for unmethylated, bisulphite converted DNA.</p></fn><fn id="t1-fn3"><label>c</label><p>Control primers not including CpG sites specific for bisulphite converted DNA.</p></fn></table-wrap-foot> <table-wrap-foot><fn id="t2-fn1"><label>a</label><p>+Indicates number of patients with a methylated tumour.</p></fn><fn id="t2-fn2"><label>b</label><p>−Indicates number of patients with no methylation detected.</p></fn></table-wrap-foot> <table-wrap-foot><fn id="t4-fn1"><p>Abbreviations: AJCC=American Joint Committee on Cancer; HR=Hazard Ratio; CI=confidence interval.</p></fn></table-wrap-foot>
<graphic xlink:href="6604452f1"/> <graphic xlink:href="6604452f2"/>
[{"mixed-citation": ["Kohonen-Corish MR, Cooper WA, Saab J, Thompson JF, Trent RJ, Millward MJ ("], "year": ["2006"], "article-title": ["Promoter hypermethylation of the O(6)-methylguanine DNA methyltransferase gene and microsatellite instability in metastatic melanoma"], "source": ["J Inv Dermatol"], "volume": ["126"], "fpage": ["167"]}]
Br J Cancer. 2008 Jul 22; 99(2):375-382