Alteration of DNA methyltransferases contributes to 5'CpG methylation and poor prognosis in lung cancer.

Overexpression of DNA methyltransferases DNMT1, DNMT3a and DNMT3b has been reported in various cancers. However, physical binding of DNA methyltransferase (DNMT) to the hypermethylated promoter of tumor suppressor genes (TSGs) has never been demonstrated in tumor tissues. In addition, alteration of DNMT at the protein level has never been reported in the same series of cancer patients. By immunohistochemical analysis, we demonstrated that DNMT1, DNMT3a and DNMT3b proteins were highly expressed in a coordinate manner in lung tumors, particularly in smokers (P=0.037, by the Fisher exact test). Patients with DNMT1 overexpression had a trend of poorer prognosis than those without such overexpression, and this prognostic significance was apparent in squamous carcinoma (SQ) patients (P=0.041, by the log-rank test). Both DNMT1 and DNMT3b overexpressions correlated with hypermethylation in the TSG promoters, especially among smoking SQ patients (P=0.012). To further explore the molecular mechanisms between altered TSGs promoter methylation and overexpression of DNMTs protein, we performed a tissue chromatin-immunoprecipitation polymerase chain reaction assay for lung tumors and showed that the methylated FHIT, p16(INK4a) and RARbeta promoters were bound by both DNMT protein and methyl-CpG-binding protein 2. These data suggest that overexpression and strong binding of various DNMTs may result in promoter hypermethylation of multiple TSGs and ultimately lead to lung tumorigenesis and poor prognosis.

Molecular diagnostic markers for lung cancer in sputum and plasma.

Lung cancer is the leading cause of cancer deaths worldwide. This study was designed to select multiple DNA markers, which have high sensitivity and specificity to serve as biomarkers for diagnosis of lung cancer. We examined the promoter hypermethylation of three tumor suppressor genes by methylation-specific PCR (MSP), and the instability of eight microsatellite markers by loss of heterozygosity (LOH) and microsatellite instability (MSI) analyses in lung tumor tissues and matched sputum specimens from 79 lung cancer patients. On the basis of the results of sensitivity, specificity, and concordance from each marker analyzed, we selected seven biomarkers, which are LOH of D9S286, D9S942, GATA49D12, and D13S170, MSI of D9S942, and methylation of p16(INK4a) and RARbeta, from the sputum analyses. These selected etiologically associated biomarkers can potentially be used as supplemental diagnostic biomarkers for early lung cancer detection.

Frequent FHIT gene loss of heterozygosity in human papillomavirus-infected non-smoking female lung cancer in Taiwan.

The fragile histidine triad (FHIT), located in chromosome region 3p14.2, had been reported to be a frequent allele with loss of heterozygosity (LOH) in smoking lung cancer and HPV-associated cervical cancer. Additionally, FHIT LOH may act as a tumor suppressor gene to involve in smoking-related lung tumorigenesis and HPV-related cervical tumorigenesis, respectively. In our previous report, a high prevalence of HPV 16/18 infection has been observed in non-smoking female lung cancer patients, and thus it was speculated that HPV 16/18 infection may increase the LOH frequency of FHIT in female cases to implicate in lung tumorigenesis. In this study, 157 lung cancer patients were enrolled and subjected to FHIT LOH analysis with three microsatellite markers. As expected, the frequency of FHIT LOH in males, smokers, and squamous cell carcinomas lung cancer patients was significantly higher than that of their corresponding counterpart (P=0.020 for gender, P<0.001 for smoking status, and P=0.038 for tumor type). Interestingly, a correlation between HPV 16 infection and FHIT LOH was observed in female lung cancer cases. To be more specifically, FHIT LOH frequency was remarkably increased from 18% (6 of 33) in HPV 16 non-infected female cases to 46% (11 of 24) in HPV 16 infected cases. The higher frequency of FHIT LOH observed in HPV 16-infected female lung tumors suggested that the involvement of HPV infection in lung tumorigenesis may, at least in part, be mediated through FHIT LOH.

Surgical results of hepatic resection for hepatocellular carcinoma with gross diaphragmatic invasion.

BACKGROUND/AIMS: Gross diaphragmatic invasion is not uncommon in patients undergoing hepatectomy for hepatocellular carcinoma. The aim of the study is to evaluate retrospectively the surgical results of hepatocellular carcinoma with gross diaphragmatic invasion undergoing en-bloc resection of diaphragm. METHODOLOGY: Between January 1989 and December 2002, 640 patients underwent curative resections for hepatocellular carcinoma in our hospital. Fifty-three patients (8.3%) who had hepatocellular carcinoma with gross diaphragmatic invasion found during operation undergoing en-bloc resection of diaphragm were assigned to group A. The other 587 patients who had hepatocellular carcinoma without gross diaphragmatic invasion were assigned to group B. The clinicopathological features, operative mortality and morbidity and long-term result of the patients between group A and B were compared. RESULTS: Of the 53 patients in group A with gross diaphragmatic invasion of hepatocellular carcinoma undergoing en-bloc resection of diaphragm, seven (13.2%) were pathologically proved to have muscular invasion of diaphragm and the other 46 (86.8%) were fibrous adhesion only or free of tumor. Primary repair of diaphragm was adequate in 52 patients (98.1%) and one required a mesh repair, Thirteen patients (24.5%) developed postoperative complication but no operative mortality occurred. There was no significant difference in operative mortality and postoperative complication rate between the two groups of patients with (group A) and without (group B) gross diaphragmatic invasion. If compared by each TNM staging (stage I, II and III) there was no significant difference between the patients of group A and B in five-year cumulative and disease-free survival. Among the 53 patients in group A, the long-term prognosis was also not significantly different between the patients with (group A1) and without (group A2) histological muscular invasion. CONCLUSIONS: En-bloc resection of diaphragm in patients with gross diaphragmatic invasion of hepatocellular carcinoma is justified since it does not significantly increase the operative mortality or postoperative complication rate and the long-term prognosis at each TNM staging is comparable to that of patients without gross diaphragmatic invasion.

Prognostic significance of intratumoral natural killer cells in primary resected esophageal squamous cell carcinoma.

BACKGROUND: Natural killer (NK) cells are important effector cells in the defense against tumors. The present study retrospectively examines the intratumoral NK cells in primary resected esophageal squamous cell carcinoma (ESCC) and the correlation between the patient' outcome and the intratumoral NK cells infiltration. METHODS: Immunohistochemistry was used to analyze the intratumoral NK cell infiltration in 38 archival specimens from patients with primary resected ESCC. RESULTS: According to the cut-off point of the staining for intratumoral NK cell infiltration, 14 (37%) cases had high level infiltration and 24 (63%) low. The 5-year survival of patients with high level NK infiltration was significantly better than that of patients with a low level of NK infiltration (p < 0.01). Multivariate analysis did not show NK cell infiltration to be a significant prognostic factor. CONCLUSIONS: Intratumoral NK cell infiltration is associated with a favorable outcome in ESCC. Intratumoral NK cell infiltration might be used as a variable with prognostic value in primary resected ESCC.

Colorectal mesenchymal tumor: a clinicopathologic study of 25 cases.

BACKGROUND: It is important to distinguish gastrointestinal stromal tumors (GISTs) from other gastrointestinal mesenchymal tumors (GIMTs), because of the malignant potential of GISTs and the availability of molecular targeted therapy. GISTs represent the most common subgroup of GIMTs, and rarely occur in the colon and rectum. The first objective of our retrospective study was to reclassify colorectal mesenchymal tumors, from files collected over 20 years, to determine if, based on immunohistologic features, the lesions were truly GISTs. The second objective was to identify the relationship between clinicopathologic features and prognostic factors of GISTs in the colon and rectum. METHODS: We evaluated all cases of colorectal mesenchymal tumor identified from the database of the Department of Surgical Pathology at Taichung Veterans General Hospital for the period 1983-2001. For 25 patients, clinical data, and information about tumor characteristics, surgical procedures, and survival outcomes, were obtained and analyzed. Histopathologic evaluations, and appropriate immunohistochemical markers, were used to distinguish between various GIMT subtypes. The relationship between KIT expression and clinicopathologic features was investigated. RESULTS: The following variables were significantly associated with different CD117 results: symptomatic presentation, location, gross features, tumor size, mitotic count, cellularity, and type of surgery. Only 18 tumors were identified as GISTs. For these, the following variables were significantly associated (by univariate analysis) with increased lethality: tumor size (p = 0.049); mitotic count (p = 0.019); nuclear atypia (p = 0.019); and tumor necrosis (p = 0.045). However, only mitotic activity showed a significant difference in the survival analysis (p = 0.0304; log-rank test). CONCLUSION: Two clinicopathologically different categories were identified from our colorectal mesenchymal tumors: intramural GISTs and polypoid submucosal leiomyomas. Our study suggests that GIST is a better categorization than smooth muscle tumor because of the malignant potential. Prognosis is strictly related to the number of mitoses. However, tumor size, nuclear atypia and tumor necrosis are probably also significant predictive factors of lethality. Future studies with DNA analysis and larger patient numbers are essential to evaluate the prognostic significance of our findings.

Ovarian serous cystadenoma with mural nodules of genital rhabdomyoma.

We present an extremely rare case of ovarian serous cystadenoma with mural nodules of rhabdomyoma. The patient, a 48-year-old woman, was admitted to our hospital with left lower abdominal pain and vaginal bleeding. A unilocular cystic tumor, measuring 13 x 10 x 10 cm, was found in her left ovary and was removed. The tumor contained clear serous fluid, approximately 600 mL, and 2 mural nodules, up to 7.5 x 5.5 x 4.5 cm. The internal cystic wall was thin for the most part and lined with ciliated cuboidal epithelium without any malignant feature. The mural part was composed of mainly more mature muscle fibers with easily discernible cross-striations, set in abundant myxoid to fibromyxoid stroma, similar to clinical and microscopic manifestations of genital rhabdomyomas reported in other sites. Because extracardiac rhabdomyoma has never been described occurring in the ovary, especially arising in serous cystadenoma, to our knowledge, this is the first case reported in the English literature.

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.

Copy number changes of target genes in chromosome 3q25.3-qter of esophageal squamous cell carcinoma: TP63 is amplified in early carcinogenesis but down-regulated as disease progressed.

AIM: By using comparative genomic hybridization, gain of 3q was found in 45-86% cases of esophageal squamous cell carcinoma (EC-SCC). Chromosome 3q25.3-qter is the minimal common region with several oncogenes found within this region. However, amplification patterns of these genes in EC-SCC have never been reported. The possible association of copy number changes of these genes with pathologic characteristics is still not clear. METHODS: Real-time quantitative PCR (Q-PCR) was performed to analyze the copy number changes of 13 candidate genes within this region in 60 primary tumors of EC-SCC, and possible association of copy number changes with pathologic characteristics was analyzed by statistics. Immunohistochemistry (IHC) study was also performed on another set of 111 primary tumors of EC-SCC to verify the association between TP63 expression change and lymph node metastasis status. RESULTS: The average copy numbers (+/-SE) per haploid genome of individual genes in 60 samples were (from centromere to telomere): SSR3: 4.19 (+/-0.69); CCNL1: 5.24 (+/-0.67); SMC4L1: 2.01 (+/-0.16); EVI1: 2.02 (+/-0.12); hTERC: 5.28 (+/-0.54); SKIL: 2.71 (+/-0.14); EIF5A2: 1.95 (+/-0.12); ECT2: 9.18 (+/-1.68); PIK3CA: 8.13 (+/-1.17); EIF4G1: 1.07 (+/-0.05); SST: 3.07 (+/-0.25); TP63: 2.51 (+/-0.22); TFRC: 2.42 (+/-0.19). Four clusters of amplification were found: SSR3 and CCLN1 at 3q25.31; hTERC and SKIL at 3q26.2; ECT2 and PIK3CA at 3q26.31-q26.32; and SST, TP63 and TFRC at 3q27.3-q29. Patients with lymph node metastasis had significantly lower copy number of TP63 in the primary tumor than those without lymph node metastasis. IHC study on tissue arrays also showed that patients with lymph node metastasis have significantly lower TP63 staining score in the primary tumor than those without lymph node metastasis. CONCLUSION: This study showed that different amplification patterns were seen among different genes within 3q25.3-qter in EC-SCC, and several novel candidate oncogenes (SSR3, SMC4L1, ECT2, and SST) were identified. TP63 is amplified in early stage of EC-SCC carcinogenesis but down-regulated in advanced stage of disease.

Frequent p16INK4a promoter hypermethylation in human papillomavirus-infected female lung cancer in Taiwan.

Inactivation of p16INK4a gene through promoter hypermethylation has been frequently observed in non small cell lung cancer; however, various studies have shown a controversial correlation between p16INK4a hypermethylation and cigarette smoking. Our recent report showed that human papillomarvirus (HPV) 16/18 infections were associated with the development of nonsmoking female lung cancer in Taiwan and we further speculated that HPV infection may be linked with p16INK4a hypermethylation. To verify the influence of environmental exposure, including cigarette smoking, environmental carcinogen exposure and HPV infections on p16INK4a hypermethylation, tumors from 162 lung patients, including 67 smoking males, 41 nonsmoking males and 58 nonsmoking females, were subjected to p16INK4a hypermethylation analysis by methylation-specific PCR. As the results showed, p16INK4a hypermethylation was detected in 40 (59.7%) of 67 smoking male, 15 (36.6%) of 41 nonsmoking male and 35 (60.3%) of 58 nonsmoking female lung tumors. This result seemed to reveal that gender and cigarette smoking both possess an equal influence on p16INK4a hypermethylation. This result also led to a speculation that HPV infection may promote p16INK4a hypermethylation in nonsmoking female lung cancer patients. From our data, p16INK4a hypermethylation frequency in nonsmoking female lung tumors with HPV infection was as high as 70% (30 of 43) compared to those without HPV infection (33%; 5 of 15). In fact, the correlation between HPV infection and p16INK4a hypermethylation was only observed in nonsmoking female lung tumors (p = 0.017), but not in smoking male or nonsmoking male lung tumors. Moreover, the reverse correlation between p16INK4a immunostaining and p16INK4a promoter hypermethylation was also only observed in nonsmoking female lung tumors. These results strongly suggested that the involvement of HPV infection in lung tumorigenesis of nonsmoking female cancer patients in Taiwan may be mediated at least in part through the increase of hypermethylation to cause p16INK4a inactivation.

Wild-type p53 overexpression and its correlation with MDM2 and p14ARF alterations: an alternative pathway to non-small-cell lung cancer.

PURPOSE: We found a relatively reduced frequency of p53 mutation with a much greater frequency of p53 protein overexpression, which reflected stabilization of p53 protein in the absence of p53 gene mutation. Therefore, we investigated the possibility of alternative mechanisms leading to p53 protein stabilization. PATIENTS AND METHODS: We performed gene and protein alteration studies on p53 and its upstream effectors, MDM2 and p14ARF, in tumors from 94 non-small-cell lung cancer (NSCLC) patients. RESULTS: Immunohistochemical and sequencing analyses indicated that 37 tumors showed overexpression of wild-type p53. An absence of nuclear staining of MDM2 protein was found in 95% of these tumors (35 of 37; P < .001). The tumors with negative MDM2 staining showed a significantly high concordance of loss of Akt activity and low MDM2 mRNA expression (P < .001). Sequencing analysis revealed five distinct MDM2 splicing variants disrupting the conserved p53 binding domain. Corresponding variant proteins were detected in three lung cancer cell lines using the Western blot analysis. Our results also indicated that among the tumors with overexpression of the wild-type p53, 92% (34 of 37) showed immunoreactivity to p14ARF (P = .001). In addition, the deregulation of p53 and MDM2 genes was significantly associated with squamous lung cancer (P < .05) and was correlated with advanced stages (P < .05) and poor prognosis (P < .05). CONCLUSION: Our data suggest that immunopositivity of p14ARF together with a low expression of MDM2 contributes to accumulation of the wild-type p53, and that deregulation of the p53-MDM2-p14ARF pathway is important in the pathogenesis and outcome of a subset of NSCLC.

Gender difference in human papillomarvirus infection for non-small cell lung cancer in Taiwan.

Our previous reports have indicated that high risk human papillomarvirus (HPV) 16/18 were much more frequently detected in lung tumors of female patients as compared to that of male patients and HPV 16/18 in lung tumors were evolutionally correlated with those in blood circulation. In the other hand, it is well known that HPV 6/11 are frequently associated with upper aerodigestive and respiratory diseases. HPV 6/11 DNA were detected in lung tumors by nested PCR and in situ hybridization to investigate if any difference in prevalent types of HPV exists between genders. Our data showed that HPV 6 infection was detected in 28.4% (40 of 141) lung tumors, which was significantly higher than that in non-cancer controls (1.7%, 1 of 60; P < 0.0001), however, such high prevalence was not observed for HPV 11. Among studied clinico-pathological parameters, HPV 6 infection was significantly related with gender (P = 0.002) and smoking status (P = 0.014). After being stratified by gender and smoking status, HPV 6 infection rate in lung tumors of non-smoking male patients was much higher than that in non-smoking female patients (33.3% versus 11.1%; P = 0.023), but no difference between smoking and non-smoking male patients (38.1% versus 33.3%). With adjustments for age, tumor type, and tumor stage, smoking male lung cancer patients had a much higher OR value (OR, 7.35; 95%CI, 2.11-25.58) for HPV 6 infection compared with 3.93 (95% CI, 1.17-13.12) of non-smoking male patients. Moreover, a higher prevalence of HPV 6 was detected in lung tumors of smoking male patients with early tumor stage than those with advanced stages (P = 0.008), but not in non-smoking male and female patients. A higher prevalence of HPV 6 in male lung cancer patients, as compared with female lung cancer patients, indicating not only different HPV infection routes for different genders, but also that HPV 6 infections may act as a prospective early risk marker of lung cancer for smoking male patients in Taiwan.

5'CpG island hypermethylation and aberrant transcript splicing both contribute to the inactivation of the FHIT gene in resected non-small cell lung cancer.

The relative contribution of promoter hypermethylation and aberrant splicing to the inactivation of the fragile histidine triad (FHIT) gene is unclear. Using genetic and epigenetic analyses, the current investigation examines the loss of protein and mRNA expression, and 5'CpG hypermethylation and allelic imbalance of the FHIT gene in a series of 129 non-small cell lung cancer (NSCLC) samples, in parallel with clinicopathological analyses. We found that 50% of NSCLC patients had aberrant protein expression, which was more frequent in squamous cell carcinomas (SQ) (69%) than in adenocarcinomas (AD) (28%) (P < 0.0001). 5'CpG hypermethylation of FHIT was identified in 31% of patients. Abnormally-sized FHIT transcripts were also observed in 24% of patients and were attributed to various exonic deletions, mainly in the region of exons 4-8. Allelic imbalance of the FHIT locus and its correlation with the status of Fhit expression, 5'CpG hypermethylation, and aberrant splicing, indicated that biallelic inactivation of Fhit expression could be induced by 5'CpG hypermethylation of one allele and alternative splicing in the other allele. Moreover, an 83% concordance in the methylation status of FHIT was demonstrated between 12 samples of bronchial precancerous lesions taken before surgery and their matched resected tumours. Our data suggest that FHIT 5'CpG hypermethylation and splicing alterations are both predominant mechanisms involved in the aberrant expression of the FHIT gene, and that FHIT 5'CpG methylation may be potentially used as a supplemental detection marker for NSCLC.

Appraisal of concomitant splenectomy in liver resection for hepatocellular carcinoma in cirrhotic patients with hypersplenic thrombocytopenia.

BACKGROUND: Liver resection usually is not recommended for hepatocellular carcinoma (HCC) in cirrhotic patients with portal hypertension. The role of concomitant splenectomy in liver resection for HCC in cirrhotic patients with hypersplenic thrombocytopenia (HT) resulting from portal hypertension remains undefined. METHODS: Among 526 cirrhotic patients who underwent liver resection for HCC, 41 underwent a concomitant splenectomy (Sp group) because of HT (platelet count

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

Expression of osteocalcin in prostate cancer before and after hormonal therapy.

BACKGROUND: Osteocalcin (OC), a noncollagenous bone matrix protein, is specifically produced by osteoblasts. Previously, we demonstrated that OC immunohistochemical staining was found in primary prostate cancer (PC) and PC metastasis to lymph node and bone. Advanced and metastatic PC is commonly treated by hormonal therapy (H/T). Inevitably, these patients will relapse and develop hormone-refractory (HR) PC. The aim of this study was to realize the OC expression in PC before H/T and after H/T when HRPC developed. MATERIALS AND METHODS: Twenty patients with PC received H/T and later developed HRPC. The interval of androgen-response was classified by good response (Group A, > or = 30 months) and poor response (Group B, < 30 months). The mean interval of androgen-response was 48.8 months in Group A (10 patients) and 17.5 months in Group B (10 patients). Formalin-fixed paraffin-embedded PC before H/T and HRPC for each patient were stained for osteocalcin. RESULTS: Osteocalcin immunohistochemical stain was detected in 18 of 20 PC before H/T and 19 of 20 of HRPC. Compared to PC before H/T, the expression of OC in HRPC increased in 5 cases (4 of Group A, 1 of Group B), did not change in 3 (2 of Group A, 1 of Group B) and decreased in 12 (4 of Group A, 8 of Group B). CONCLUSION: Our data suggest the expression of OC in PC is not correlated to the response of H/T. OC expression was still noted in 95% of HRPC. OC may be a target for treatment of HRPC.

The correlation between aberrant connexin 43 mRNA expression induced by promoter methylation and nodal micrometastasis in non-small cell lung cancer.

Reduced connexin (Cx) 43 gene expression has been shown in most of lung tumors and cancer cell lines. Although aberrant Cx43 gene expression was linked with lung tumorigenesis, our understanding to the mechanism was still limited. We hypothesized that the evidence of aberrant Cx43 gene expression was gradually intensified from adjacent normal lung tissues surrounding tumors toward tumor tissues. In this study, 90 lung tumors and adjacent normal tissues were collected to examine Cx43 mRNA expression by reverse transcription-PCR (RT-PCR). Our data showed that Cx43 mRNA expression in adjacent normal lung tissue was significantly correlated with nodal involvement (P = 0.03), but the similar trend was not observed in tumor tissues. To verify whether lack of Cx43 mRNA expression resulted from promoter methylation, PCR-based methylation assay was performed for Cx43 promoter methylation analysis. A higher frequency of promoter methylation was observed in Cx43 mRNA-negative patients (21 of 33, 63.7%) compared with Cx43 mRNA-positive patients (3 of 57, 5.3%, P < 0.0001). To elucidate whether aberrant Cx43 gene expression originated from adjacent normal lung tissues, 25 lung tumors and each of five adjacent normal tissues at various distances from tumor tissues were collected to examine Cx43 mRNA and protein expression by RT-PCR and Western blot, respectively. The results show that Cx43 mRNA and protein expressions gradually decreased from adjacent normal lung tissues to tumor tissues with a positive correlation to the distance from the tumor tissues. Gel-shift assay data also revealed that shifted band binding with AP1 was only observed in adjacent normal tissues, which were far from the tumor tissues. These results indicate that promoter methylation may interfere with AP1 binding to the promoter to cause aberrant Cx43 gene expression. Thus, Cx43 mRNA in adjacent normal tissue surrounding lung tumor simply detected by RT-PCR may act as a molecular marker of nodal micrometastasis in non-small cell lung cancer.

Loss of Smad4 protein expression occurs infrequently in endometrial carcinomas.

Smad4 is a member of the Smad proteins, which are needed for mediating signals of transforming growth factor beta from the cell surface to the nucleus. Smad4 is also a tumor suppressor gene for cancers of the pancreas, colon, and lung. The aim of this study was to investigate the expression and prognostic significance of this gene product in endometrial cancer. Immunohistochemical staining for Smad4 was performed on formalin-fixed, paraffin-embedded specimens of endometrial tumors with an anti-Smad4 monoclonal antibody (clone B8): 97 primary endometrial carcinomas, 20 cases of endometrial hyperplasia, and 26 cases of metastases from endometrial carcinoma. The immunoreactivity of each tumor was correlated with the clinical and histopathologic parameters of the patients. Diffusely positive expression of Smad4 protein was detected in all 20 cases of endometrial hyperplasia and in most of the primary and metastatic endometrial cancers. The frequency of positive expression decreased progressively with tumor grade. Clinically, however, it was not associated with tumor progression, nor did it predict patient outcome. Although loss of heterozygosity at chromosome 18q21 (the location of the Smad4 gene) is frequent in endometrial carcinomas, the authors show in this immunohistochemical study that inactivation of this gene occurs infrequently in this tumor.