[Suggesting the Significance of Pericardial Fat Pad in Bronchial Stump Fistula].

Bronchial stump fistula is a post-operative complication with poor outcome after pulmonary lobectomy. In order to prevent this complication, the bronchial stump is covered with pericardial fat tissue in our hospital. The case was 58 year old male who received adjuvant chemotherapy after sigmoidectomy for sigmoid colon cancer. As he developed multiple pulmonary metastases, 48 courses of chemotherapy were performed. The lesions had been localized at the right lower lobe, and neither increase in the size of these lesions nor development of other lesions were observed. Hence, an operation was performed. After right lower lobectomy, the bronchial stump was covered with the pericardial fat tissue. Three months after the operation, he developed pneumothorax, and bubbles were detected inside the fat. The pneumothorax was cured conservatively, and the bubbles disappeared spontaneously after 10 months. It is rare that the patient with bubbles in the covering tissue observed for a long time is cured conservatively, suggesting the significance of the stump pad.

A phase II study of weekly paclitaxel as second-line chemotherapy for advanced gastric Cancer (CCOG0302 study).

BACKGROUND: Although paclitaxel was given triweekly in phase II trials prior to its approval for gastric cancer in Japan, it is currently more often delivered by a weekly schedule in the second-line setting. PATIENTS AND METHODS: A phase II trial with response rate as the primary end-point was conducted. Patients with metastatic or unresectable gastric adenocarcinoma who had measurable lesions and had disease progression with the front-line chemotherapy were treated by weekly administration of paclitaxel at a dose of 80 mg/m2. RESULTS: Forty-five patients were accrued and 44 were assessable for response. Partial responses were observed in 7 patients (16%). Stable disease was documented in further 14 patients (48%). Median progression-free survival of all patients enrolled was 2.6 months and median overall survival was 7.8 months. Toxicity was mild and manageable, the most frequent > or = grade 3 toxicity being neutropenia occurring in 16% of the patients. CONCLUSION: With modest response rate, favorable toxicity profile, and progression-free or overall survival similar to those of more intense combination regimens, weekly paclitaxel remains a rational therapeutic option for gastric cancer refractory to the first-line chemotherapy.

Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling.

BACKGROUND: Vascular endothelial growth factor C (VEGF-C) stimulates tumor lymphangiogenesis (i.e., formation of lymphatic vessels) and metastasis to regional lymph nodes by interacting with VEGF receptor 3 (VEGFR-3). We sought to determine whether inhibiting VEGFR-3 signaling, and thus tumor lymphangiogenesis, would inhibit tumor metastasis. METHODS: We used the highly metastatic human lung cancer cell line NCI-H460-LNM35 (LNM35) and its parental line NCI-H460-N15 (N15) with low metastatic capacity. We inserted genes by transfection and established a stable N15 cell line secreting VEGF-C and a LNM35 cell line secreting the soluble fusion protein VEGF receptor 3-immunoglobulin (VEGFR-3-Ig, which binds VEGF-C and inhibits VEGFR-3 signaling). Control lines were transfected with mock vectors. Tumor cells were implanted subcutaneously into severe combined immunodeficient mice (n = 6 in each group), and tumors and metastases were examined 6 weeks later. In another approach, recombinant adenoviruses expressing VEGFR-3-Ig (AdR3-Ig) or beta-galactosidase (AdLacZ) were injected intravenously into LNM35 tumor-bearing mice (n = 14 and 7, respectively). RESULTS: LNM35 cells expressed higher levels of VEGF-C RNA and protein than did N15 cells. Xenograft mock vector-transfected LNM35 tumors showed more intratumoral lymphatic vessels (15.3 vessels per grid; 95% confidence interval [CI] = 13.3 to 17.4) and more metastases in draining lymph nodes (12 of 12) than VEGFR-3-Ig-transfected LNM35 tumors (4.1 vessels per grid; 95% CI = 3.4 to 4.7; P<.001, two-sided t test; and four lymph nodes with metastases of 12 lymph nodes examined). Lymph node metastasis was also inhibited in AdR3-Ig-treated mice (AdR3-Ig = 0 of 28 lymph nodes; AdLacZ = 11 of 14 lymph nodes). However, metastasis to the lungs occurred in all mice, suggesting that LNM35 cells can also spread via other mechanisms. N15 tumors overexpressing VEGF-C contained more lymphatic vessels than vector-transfected tumors but did not have increased metastatic ability. CONCLUSIONS: Lymph node metastasis appears to be regulated by additional factors besides VEGF-C. Inhibition of VEGFR-3 signaling can suppress tumor lymphangiogenesis and metastasis to regional lymph nodes but not to lungs.

[Two cases of advanced gastric cancer with peritoneal dissemination acquired long-term response three years or more by postoperative treatment with oral anticancer drug TS-1].

The first patient is a 60-year-old man who underwent total gastrectomy and splenectomy for type-3 gastric cancer after neoadjuvant chemotherapy. The second patient is a 69-year-old woman who underwent distal gastrectomy for type-2 gastric cancer and pyloric stenosis after neoadjuvant chemotherapy. Some peritoneal dissemination was observed in these two cases. No re-growth of peritoneal dissemination was seen for three years or more from treatment with the oral anticancer drug TS-1. Treatment on an outpatient basis, therefore, greatly contributed to their quality of life. We consider TS-1 as a first-line anti-cancer drug for advanced gastric cancer.

Significant up-regulation of a novel gene, CLCP1, in a highly metastatic lung cancer subline as well as in lung cancers in vivo.

Most lung cancer patients are unfortunately uncurable and die because of widespread metastases, thus indicating the importance of identification of molecules with a crucial role in this process. Our previous expression profiling analysis of a highly metastatic lung cancer cell line, NCI-H460-LNM35, and its parental low metastatic line, NCI-H460-N15, revealed significant up-regulation of both known and unknown genes in LNM35. In this study, we describe the isolation and detailed characterizations of a novel gene, named CLCP1, which corresponds to one of such expression sequence tags with up-regulated expression in LNM35. The CLCP1 gene was found to encode a protein with 775 amino acids with structural similarities to, but distinct from neuropilins, cell surface receptors for VEGF165 and semaphorins. Notably, CLCP1 was shown to be up-regulated not only in LNM35 in association with its acquisition of metastatic phenotype during in vivo selection, but also in a significant fraction of lung cancers in vivo with high frequency in metastatic lesions, warranting future studies for a better understanding of the molecular mechanisms of lung cancer metastasis.

Gene expression-based, individualized outcome prediction for surgically treated lung cancer patients.

Individualized outcome prediction classifiers were successfully constructed through expression profiling of a total of 8644 genes in 50 non-small-cell lung cancer (NSCLC) cases, which had been consecutively operated on within a defined short period of time and followed up for more than 5 years. The resultant classifier of NSCLCs yielded 82% accuracy for forecasting survival or death 5 years after surgery of a given patient. In addition, since two major histologic classes may differ in terms of outcome-related expression signatures, histologic-type-specific outcome classifiers were also constructed. The resultant highly predictive classifiers, designed specifically for nonsquamous cell carcinomas, showed a prediction accuracy of more than 90% independent of disease stage. In addition to the presence of heterogeneities in adenocarcinomas, our unsupervised hierarchical clustering analysis revealed for the first time the existence of clinicopathologically relevant subclasses of squamous cell carcinomas with marked differences in their invasive growth and prognosis. This finding clearly suggests that NSCLCs comprise distinct subclasses with considerable heterogeneities even within one histologic type. Overall, these findings should advance not only our understanding of the biology of lung cancer but also our ability to individualize postoperative therapies based on the predicted outcome.

Expression of CD109 in human cancer.

It was recently reported that the human CD109 gene encodes a glycosyl-phosphatidylinositol-anchored glycoprotein that is a member of the alpha(2)-macroglobulin/C3, C4, C5 family of thioester-containing proteins. In this study, we found that the expression of mouse CD109 gene was upregulated in NIH3T3 cells expressing RET tyrosine kinase with a multiple endocrine neoplasia 2B mutation. Northern blot analysis showed a high level of expression of the CD109 gene only in the testis in normal human and mouse tissues. In addition, its expression was high in some human tumor cell lines, which included squamous cell carcinoma and glioblastoma cell lines, whereas it was undetectable in neuroblastoma and small-cell lung carcinoma cell lines. When CD109 expression was examined in 33 cases of human lung cell carcinomas by quantitative RT-PCR, a significant high expression of CD109 was detected in about half of squamous cell carcinomas examined, but not in adenocarcinoma, large-cell carcinoma and small-cell carcinoma. Similarly, upregulation of CD109 was observed in nine out of 17 esophageal squamous cell carcinomas. Thus, these results suggested that CD109 might be a useful molecular target for the development of new therapeutics for malignant tumors, such as squamous cell carcinoma.

Mitochondrial D-loop mutations as clonal markers in multicentric hepatocellular carcinoma and plasma.

PURPOSE: Hepatocellular carcinoma (HCC) is a highly malignant tumor prone to multicentric occurrence. Differentiation between a true relapse of HCC and a second primary tumor is of clinical importance. We sought to identify mitochondrial mutations in HCC and test their use as clonal markers in this disease. EXPERIMENTAL DESIGN: Primary HCC tissue samples were obtained from 19 patients and analyzed for mutations within the mitochondrial displacement loop (D-loop). The discovered mutations were used to determine tumor clonality and provided the basis for detection of tumor DNA in corresponding plasma samples. RESULTS: Thirteen of 19 HCC cases (68%) were identified as having D-loop mitochondrial DNA (mtDNA) mutations in at least one tumor. In 3 of these 13 cases, the same mutation was observed in multiple tumors, indicating monoclonal origin. Remarkably, in 8 of 13 mutated cases, we detected deletion/insertion mutations in the C-tract, a recently reported hotspot and potential replication start site of the closed, circular mitochondrial genome. In addition, we detected mutant mtDNA in 8 of 10 tested paired plasma DNA samples using a highly sensitivity molecular assay. CONCLUSIONS: mtDNA mutations within the D-loop control region are a frequent event in HCC, providing a molecular tool for the determination of clonality. In addition, detection of tumor-specific mtDNA mutations in plasma DNA needs to be explored further for monitoring patients with primary HCC.

Allelic imbalance at 1p36 in the pathogenesis of human hepatocellular carcinoma.

BACKGROUND/AIMS: It has been well accepted that there is an allelic imbalance at a subtelomeric region of chromosome 1p in various human malignancies including hepatocellular carcinoma. We conducted a detailed deletion mapping study at 1p36 loci in 65 hepatocellular carcinomas as an initial step towards positional cloning of the target gene. METHODOLOGY: We performed a fine-scale deletion mapping using 10 highly polymorphic microsatellite markers along the telomeric region of 1p (1p32-p36.3) and compared the clinicopathologic features with allelic imbalance of 1p36 loci. RESULTS: Allelic imbalance occurred in at least one locus on 1p in 32 (49.2%) of the 65 cases. Consequently, it was confirmed that a common region of overlaps was restricted to 1p36 and it was further clearly demonstrated that there were at least three independent regions in 1p36 (1p36.1, p36.2'36.3 and p36.3). An apparent trend towards significance has been demonstrated between allelic imbalance at 1p36 loci and tumors with classifcation T1-T2 (p<0.01). CONCLUSIONS: The present study demonstrated that the shortest region of overlap was confined within 1p36 chromosomal sub-band in human hepatocellular carcinoma, which might be involved in an early step of hepatocarcinogenesis, and it encourages future studies to identify the putative tumor suppressor gene(s) at 1p36.

Overexpression of pituitary tumor transforming gene 1 in HCC is associated with angiogenesis and poor prognosis.

The pituitary tumor transforming gene 1 (PTTG1) protein is cell-cycle regulated and is identified as a human securin that inhibits sister chromatid separation and is involved in transformation and tumorigenesis. PTTG1 has very low or undetectable expression in most normal human tissues, but it is abundantly expressed in malignant cell lines and pituitary tumors. In this study, we investigated human PTTG1 expression in 62 hepatocellular carcinoma (HCC) specimens using quantitative real-time reverse transcription polymerase chain reaction analysis. We found that, compared with corresponding noncancerous liver tissues, PTTG1 was remarkably overexpressed in HCCs (PTTG1/beta-actin; 0.443 +/- 0.073 vs. 0.068 +/- 0.007; P < .0001). Furthermore, we found a significant correlation between PTTG1 expression and serum alpha-fetoprotein level (P < .001). Univariate and multivariate analyses revealed that the PTTG1 messenger RNA (mRNA) expression was an independent prognostic factor for disease-free (odds ratio 2.70; P = .037) and overall (odds ratio 5.35; P = .007) survival. Moreover, we discovered a significant relationship between PTTG1 expression and intratumoral microvessel density. Our data supported an important role for PTTG1-mediated upregulation of fibroblast growth factor (FGF)-2, one of angiogenesis and modulation of tumor progression, in hepatocarcinogenesis. In conclusion, PTTG1 might be critically involved in the development of HCCs through the promotion of angiogenesis. PTTG1 is overexpressed in HCC and our results suggest that PTTG1 mRNA expression has prognostic significance for the survival of postoperative patients with HCC.

Decreased expression and frequent allelic inactivation of the RUNX3 gene at 1p36 in human hepatocellular carcinoma.

BACKGROUND/AIMS: Alteration in transforming growth factor-beta signaling pathway is one of the main causes of hepatocellular carcinoma (HCC). The human runt-related transcription factor 3 gene (RUNX3) is an important component of this pathway. RUNX3 locus 1p36 is commonly deleted in a variety of human cancers, including HCC. Therefore, we examined genetic and epigenetic alterations of RUNX3 in human HCC. METHODS: Five HCC cell lines and 41 patients with HCC were investigated in this study. We examined the expression of RUNX3 mRNA, methylation status of RUNX3 promoter region, loss of heterozygosity (LOH) at 1p36, and mutation analysis. These results were compared with clinicopathological data. RESULTS: Promoter hypermethylation was detected in four (80%) of five HCC cell lines and 31 (75.6%) of 41 HCC tissues, confirmed by sequence of bisulfite-treated DNA. LOH was detected in 14 (37.8%) of 37 HCC. By comparison with clinicopathological data, hypermethylation was more common in hepatitis C virus antibody and formation of capsule-positive cases, and decrease of expression was correlated strongly with advanced stage and LOH-detected cases. CONCLUSION: Hypermethylation and LOH appear to be common mechanisms for inactivation of RUNX3 in HCC. Therefore, RUNX3 may be an important tumor suppressor gene related to hepatocarcinogenesis.

Frequent promoter methylation and gene silencing of CDH13 in pancreatic cancer.

It has recently been reported that CDH13 expression is silenced by aberrant methylation of the promoter region in several cancers. We examined the methylation status of the CDH13 gene in pancreatic cancer using methylation-specific PCR (MSP), and detected aberrant methylation of CDH13 in all 6 pancreatic cancer cell lines examined. To confirm the status of the CDH13 gene in relation to the methylation pattern, we next examined CDH13 expression in these cell lines using reverse transcription (RT)-PCR. As expected, no CDH13 expression was detected in any of the 6 pancreatic cancer cell lines. Moreover, 5-aza-2'-deoxycytidine (5-aza-dC) treatment of CDH13-methylated cell lines led to restoration of CDH13 expression. Among primary pancreatic cancers, 19 of 33 (58%) cases exhibited CDH13 methylation, while no cases exhibited it in corresponding normal pancreatic tissues. CDH13 methylation was detected even in relatively early pancreatic cancers, such as stage II cancers and cancers less than 2 cm in diameter. Our results suggest that the aberrant methylation of CDH13 occurs frequently in pancreatic cancer, even at a relatively early stage.

Focal adhesion kinase is overexpressed in hepatocellular carcinoma and can be served as an independent prognostic factor.

BACKGROUND/AIMS: The development of human malignancies can be attributed to aberrant regulation of intracellular signal transduction pathways. In the current study, we aimed to evaluate focal adhesion kinase (FAK), a non-receptor tyrosine kinase, expression in hepatocellular carcinoma (HCC), and to explore the prognostic significance of FAK. METHODS: We investigated FAK mRNA expression in 60 HCC specimens using quantitative real-time reverse transcription polymerase chain reaction analysis, and the correlation between FAK expression and clinicopathologic parameters. FAK protein expression was examined using Western blot analysis and an immunohistochemical study. RESULTS: We found that FAK mRNA was overexpressed in HCCs compared with the corresponding non-cancerous liver tissues (P=0.0008). The FAK overexpression correlated significantly with tumor size (P=0.034) and serum AFP level (P=0.030). Univariate and multivariate analyses revealed that FAK mRNA expression was an independent prognostic factor for disease-free (risk ratio 3.83; P=0.024) and overall (risk ratio 7.14; P=0.015) survival. Besides, we confirmed immunohistochemically that the FAK protein was detectable in cancer cells despite non-expression in corresponding non-cancerous tissues. CONCLUSIONS: Our results suggest that FAK mRNA expression has prognostic significance for the survival of patients with HCC.

A novel target gene, SKP2, within the 5p13 amplicon that is frequently detected in small cell lung cancers.

We investigated DNA copy-number aberrations in 22 cell lines derived from small cell lung cancers (SCLCs) using comparative genomic hybridization. A minimal common region at 5p13, within the 5p11-p13 amplicon that was most frequently involved, harbored the CDH6, PC4, and SKP2 genes. These three genes showed amplification and consequent overexpression in the SCLC cell lines. SKP2 positively regulates progression of cell cycle by targeting several regulators, such as the cell-cycle inhibitor p27(KIP1), for ubiquitin-mediated degradation. SKP2 was amplified in 7 (44%) of 16 primary SCLC tumors, and consequently overexpressed in 10 (83%) of the 12 of those tumors we examined. Expression levels of SKP2 protein were cell cycle-dependent in SCLC cells as well as in normal cells, and were correlated with the DNA copy-number of the gene. There was an inverse correlation between the expression of SKP2 and p27(KIP1) proteins. Down-regulation of SKP2 using an anti-sense oligonucleotide remarkably suppressed the growth of SCLC cells. Our results indicate that SKP2 is likely to be a target of the 5p13 amplification and to play an important role in the growth of SCLC cells.