The induction of MIG6 under hypoxic conditions is critical for dormancy in primary cultured lung cancer cells with activating EGFR mutations.

The biologic activity of individual cancer cells is highly heterogeneous. Hypoxia, one of the prominent features of a tumor microenvironment, is thought to be causal in generating this cellular heterogeneity. In this study, we revealed that primary lung cancer cells harboring activating epidermal growth factor receptor (EGFR) mutations generally entered a dormant state when hypoxic. We found that heterodimer formation of the ERBB family receptor tyrosine kinases (RTKs), and their subsequent downstream signaling, was diminished under hypoxic conditions, although phosphorylation of the EGFR was retained. Dormant lung cancer cells were found to be resistant to EGFR tyrosine kinase inhibitor (TKI) treatment. In terms of mechanism, we found that a negative regulator of ERBB signaling, MIG6/ERRFI1/RALT/Gene33, was induced by hypoxia both in vitro and in vivo. MIG6 expression prevented heterodimer formation of ERBB family RTKs, and suppressed their downstream signaling. Knockdown of MIG6 enhanced tumor cell growth under hypoxic conditions, and promoted the phosphorylation of ERK and AKT via increased EGFR-HER3 binding. Critically, sensitivity to an EGFR-TKI, as well as to irradiation under hypoxic conditions, was increased in MIG6 knockdown cells. The expression of MIG6 was partly correlated with a pS6 negative zone in patient tumors. Analyses of tumor sections from 68 patients with activating EGFR mutations showed that patients with high MIG6 expression showed significantly shorter survival after EGFR-TKI treatment than other groups. Collectively, our data suggest that dormant cancer cells with a high MIG6 expression level might be one of the causes of EGFR-TKI resistance in EGFR mutant lung cancer cells.

Novel prognostic prediction models for patients with stage IV colorectal cancer after concurrent curative resection.

BACKGROUND: We developed a prediction tool for recurrence and survival in patients with stage IV colorectal cancer (CRC) following surgically curative resection. PATIENTS AND METHODS: From January 1983 to December 2012, 113 patients with CRC and synchronous liver and/or lung metastatic CRC were investigated at the Osaka Medical Center for Cancer and Cardiovascular Diseases. All patients underwent curative resection of primary and metastatic lesions. In the group of patients who underwent surgery from 1983 to 2008, a Cox regression model was used to develop prediction models for 1-year, 3-year and 5-year cancer-specific survival (CSS) and relapse-free survival (RFS). In the other group of patients who underwent surgery from 2009 to 2012, the developed prediction model was validated. RESULTS: Univariate analysis of clinicopathological factors showed that the following factors were significantly correlated with CSS and RFS: preoperative serum carcinoembryonic antigen level, tumour location, pathologically defined tumour invasion and lymph node metastasis, and synchronous metastatic lesions. Using these variables, novel prediction models predicting CSS and RFS were constructed using the Cox regression model with concordance indexes of 0.802 for CSS and 0.631 for RFS. The prediction models were validated by external data sets in an independent patient group. CONCLUSIONS: We developed novel and reliable personalised prognostic models, integrating tumour, node, metastasis (TNM) factors as well as the preoperative serum carcinoembryonic antigen level, tumour location and metastatic lesions, to predict patients' prognosis following surgically curative resection. This individualised prediction model may help clinicians in the treatment of postoperative stage IV CRC following surgically curative resection.

Role of combined 18F-FDG-PET/CT for predicting the WHO malignancy grade of thymic epithelial tumors: a multicenter analysis.

INTRODUCTION: To investigate the performance of combined (18)F-FDG-PET/CT as a predictor of the WHO-classification based malignancy grade in thymic epithelial tumors. METHODS: From 05/06 to 02/12, the data of 47 patients with thymic epithelial tumors assessed by (18)F-FDG-PET/CT before being surgically treated were collected in 3 centers and retrospectively reviewed for the purposes of this study. The SUVmax and the SUVmax/T index (the ratio tumor-SUVmax to tumor-size) have been matched with specific subgroups of the WHO-classification: low-risk thymomas (types A-AB-B1), high-risk thymomas (types B2-B3) and thymic carcinomas (type C). RESULTS: There were 22 men and 25 women (age range: 31-84 yrs). Mean tumor size was 44.7 ± 19.0 mm. The WHO-classification was: type-A #2, type-AB #11, type-B1 #9, type-B2 #9, type-B3 #9 and type-C #7. The SUVmax and the SUVmax/T were found to be predictive factors useful to distinguish thymomas from thymic carcinomas (SUVmax: area under ROC-curve: 0.955, p = 0.0045; SUVmax/T-size: area under ROC-curve: 0.927, p = 0.0022). Moreover, both parameters were found to be correlated with the WHO malignancy grade (low-risk thymomas; high-risk thymomas; thymic carcinoma), Spearman correlation coefficients being 0.56 (p < 0.0001) and 0.76 (p < 0.0001), respectively for the SUVmax and for the SUVmax/T index. In addition, the SUVmax is also significantly correlated with Masaoka stage (Spearman correlation coefficient: 0.30, p = 0.0436) CONCLUSIONS: A significant relationship was observed between (18)F-FDG-PET/CT findings and histologic WHO-classification for this cohort of thymic epithelial tumors. Thus, on the basis of these evidences, we infer that (18)F-FDG-PET/CT may be useful to predict histology and the WHO classes of risk.

[Complete resection of Pancoast tumor following induction chemoradiotherapy improves survival].

OBJECTIVE: Pancoast tumors are some of the most challenging thoracic malignant diseases to treat because of their proximity to vital structures at the thoracic inlet. We retrospectively analyzed 23 patients with pT3-4, N0-3 Pancoast tumors who underwent combined chest wall resection including the 1st rib, and discuss the anatomical considerations, assessment of induction therapy, and surgical approaches for these cancers. METHODS: Between 1983 and 2006, 23 patients with Pancoast tumors underwent combined resection of the 1st rib at our institute. Of those, 21 were male and 2 were female, with an average age of 58 years. There were 10 each of squamous cell carcinoma and adenocarcinoma, 2 large cell carcinoma, and 1 adenosquamous carcinoma. Over the past decade, induction chemoradiotherapy (>40Gy) was employed before surgery. RESULTS: A posterior approach was employed in 14 patients, an anterior approach in 7, and a combined anterior and posterior approach in 2. Sixteen patients underwent complete resection. One of 7 patients undergoing incomplete resection (4.3%) died on the 45th postoperative day. The 3- and 5-year survival rates were 50 and 22%, respectively, for patients with complete resection. No case survived for more than 8 months out of the 7 patients with incomplete resection. Fourteen patients with pN0 showed significantly better survival than those with pN1-3 (p = 0.0053). CONCLUSION: Recent literature and our results suggest that patients with pN0 and/or a pathological complete response (pCR) after induction chemoradiotherapy could achieve long-term survival after complete resection.

[Surgical treatment for the second double lung cancers following chemotherapy for small cell lung cancer].

We report a case with surgery for the 2nd primary double lung cancers-adenocarcinoma and squamous cell carcinoma which developed in the right upper lobe after 5 years successful control by chemotherapy for small cell lung cancer in the left upper lobe. Long term survivors with small cell lung cancer have recently increased as a result of progress of chemotherapy. Therefore, 2nd primary lung cancer is not rare after the treatment for the initial small cell lung cancer. Although several causes have been proposed on the development of 2nd primary lung cancer after small cell lung cancer treatment, smoking history was strongly suggested as a cause in this case. Careful follow-up especially focusing on 2nd primary lung cancer development is necessary for patients after successful treatment for small cell lung cancer.

[Extrapleural pneumonectomy for relapsed solitary fibrous tumors of the pleura with pleural dissemination].

We report a case of a 62-year-old female with a prior thoracotomy for solitary fibrous tumor of the diaphragmatic pleura. There was no clear evidence of malignant solitary fibrous tumor of the pleura (SFTP). In the 19th postoperative month, she had a disseminated recurrence of SFTP in the left thoracic cavity. There was no evidence of metastasis from medical imaging. Accordingly, a left extrapleural pneumonectomy was performed. Pathological examination revealed a disseminated recurrence of malignant SFTP, showing a higher grade of malignancy, because the resected specimen was identical to the only section suspicious of malignancy in the previous tumor. She had no complaint and kept better performance status until the 7th postoperative month after the re-resection, when she had a recurrence in the left thoracic cavity and dissemination in the peritoneal cavity. She died of the recurrence 15 months after the re-resection and 34 months after the prior thoracotomy.

An immunohistochemical study of cyclooxygenase (COX)-2 expression in endocrine tumors of the pancreas.

Cyclooxygenase (COX) is a key rate-limiting enzyme in prostaglandin biosynthesis. There are two isoforms of COX, referred to as COX-1 and COX-2. COX-2, an inducible form of COX, is found to be overexpressed in various neoplasms and is believed to play an important role in tumorigenesis and tumor development. In this study, we investigated expression of the COX-2 protein in human endocrine tumors of the pancreas (N=23; 6 insulinomas, one glucagnoma, 2 gastrinomas, and 14 non-functioning tumors) using immunohistochemistry. Strong COX-2 expression was confirmed in normal islet tissue as previously reported. COX-2 immunoreactivity was detected in 65% (15 out of 23) of these tumors with a moderate to strong intensity. In all nine functioning tumors, COX-2 expressions were preserved with the weak or strong intensity. In contrast, COX-2 was present in 6 out of 14 nonfunctioning tumors. The correlation between COX-2 expression and their function was significant (p<0.05). We found that expression of this enzyme was detected in 11 out of 15 benign tumors and in 4 out of 8 malignant tumors, respectively. Our results suggest that COX-2 may play an important role in the endocrine function of islet tumors. Additionally, malignancy was not related to COX-2 expression.

Differential expression of cyclooxygenase-2 (COX-2) in human bile duct epithelial cells and bile duct neoplasm.

It is well known that chronic inflammatory conditions involving the bile ducts predispose to the development of bile duct carcinoma, although the relationship between chronic inflammation and malignant transformation is unclear. In this study, by combining immunohistochemistry and computer imaging techniques, we quantified and compared the cyclooxygenase-2 (COX-2) protein expression levels of epithelial cells according with their histopathological backgrounds. This technique revealed that the highest levels of COX-2 were expressed in bile duct carcinoma cells, mainly in cytoplasm, and the expression pattern was homogenous and abundant. Moderate levels of COX-2 protein expression were also observed in noncancerous epithelial cells with inflammatory reaction, but the staining intensity was heterogeneous among the positive cells exhibiting inflammation. In contrast, only scattered weak reactivity of COX-2 protein was observed in the noncancerous bile duct epithelial cells without inflammatory reaction. Moreover, bile duct epithelial cells in primary sclerosing cholangitis (PSC) showed very strong expression of COX-2 protein, that was comparable with carcinoma cells. On the other hand, primary biliary cirrhosis (PBC) epithelial cells showed moderate levels of COX-2 expression. In addition, specific COX-2 inhibitors, JTE-522 and NS-398, directly inhibited the growth of 4 bile duct carcinoma and 1 gall bladder carcinoma cell lines that expressed COX-2 protein, in vitro. These data suggest that COX-2 expression might regulate carcinogenesis of bile duct epithelial cells in inflammatory regions and tumor progression in this cancer. The data also suggest that COX-2 selective inhibitors might have therapeutic effects not only on bile duct carcinoma, but other hepatobiliary carcinomas.

Human stomach-specific gene, CA11, is down-regulated in gastric cancer.

To reveal the implication in gastric cancer pathogenesis of the novel human gene referred to as CA11, which was recently isolated by a differential display technique using normal gastric mucosa and gastric cancer tissue, we examined CA11 expression in 50 primary gastric cancers and also introduced the CA11 gene into gastric cancer cells. RNA dot blot analysis against various human organs and developmental stages demonstrated that CA11 was intensively expressed especially in normal stomach tissue. Northern blot analysis showed that expression of the CA11 gene in cancer tissue was down-regulated compared with normal tissue. Semi-quantitative RT-PCR also demonstrated that CA11 gene expression was decreased in 41 out of 50 (82%) of the gastric cancer tissues, when compared with normal stomach tissues, while no relationship was found between CA11 expression and various clinicopathological characteristics including histological type, depth of invasion, lymph node metastasis, and clinical stage. Immunohistochemical analysis with anti CA11 antibody showed that CA11-positive staining was observed in the surface regions of normal gastric epithelium, but was found faintly or not at all in cancer tissues. CA11 transfected MKN28 cells also displayed a marked decrease in the number of colony formations when compared to double normal controls. These findings suggest that the loss of CA11 expression in gastric tissues may play an important role in gastric carcinogenesis.

Genetic detection for micrometastasis in lymph node of biliary tract carcinoma.

The presence of regional lymph node metastasis is one of the most significant poor-prognosis factors in patients with biliary tract carcinoma. To establish a sensitive reverse transcription (RT)-PCR assay to detect micrometastases in lymph nodes of biliary tract carcinoma, we first investigated the optimal markers in biliary tract carcinoma. The expressions of the six candidates for a suitable RT-PCR marker [mammaglobin B, carcinoembryonic antigen (CEA), cytokeratin (CK) 20, prostate-specific antigen, and melanoma antigens (MAGE-1 and MAGE-3)] were evaluated in two bile duct cancer cell lines and human biliary tract carcinoma tissues. Of 32 carcinoma tissues, mammaglobin B, CEA, prostate-specific antigen, MAGE-1, MAGE-3, and CK 20 were expressed in 28 (88%), 26 (81%), 4 (13%), 5 (16%), 7 (22%), and 9 (28%), respectively. Mammaglobin B and CEA were considered to be good markers of the six candidates. We then examined 209 lymph nodes obtained from 15 patients with biliary tract carcinoma by RT-PCR assay using both mammaglobin B and CEA and compared the results with those of histological examination. All of 20 histologically positive lymph nodes for metastasis displayed the PCR product(s) of marker genes. Of 189 histologically negative nodes, 24 (13%) nodes expressed mammaglobin B and/or CEA mRNA, suggesting the presence of micrometastasis. Our findings suggest that mammaglobin B and CEA could be useful RT-PCR markers for the detection of lymph node micrometastases in biliary tract carcinomas. Our RT-PCR assay allows accurate clinical staging necessary for patient stratification with respect to adjuvant therapy after surgery.

Outcome of pancreatic cancer patients based on genetic lymph node staging.

We prospectively assessed the clinical value of genetic staging of lymph node metastasis in patients with pancreatic adenocarcinoma who underwent curative surgery. K-ras gene mutations were detected in the primary tumors in 18 of 25 patients with pancreatic adenocarcinoma. Among these 18 patients, mutated K-ras gene was also found in at least one lymph node in 13 patients. Of these 13 patients, seven had no evidence of histological nodal involvement and six had histological lymph node metastasis. Although there was no significant difference in overall survival rates between the pathological node-negative and -positive patients, overall survival of the five patients with nodes-negative for the mutated K-ras gene were significantly better than that of the 13 patients with genetically metastasis-positive nodes (p<0.001). Furthermore, overall survival of the six patients with genetically metastasis-positive nodes limited to peripancreatic area was significantly better than that of seven patients with genetical metastasis in lymph nodes beyond the peripancreatic areas (p=0.018). These findings suggest that detection of K-ras gene mutations in lymph nodes may be clinically useful to assess the accurate tumor staging and to stratify the patient with pancreatic adenocarcinoma who are at high or low risk for recurrence after curative surgery.

Up-regulation of cyclooxygenase-2 in squamous carcinogenesis of the esophagus.

Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies including squamous cell carcinomas (SCCs) of the esophagus, but little is known about COX-2 expression in premalignant esophageal squamous dysplasia. To elucidate the role of COX-2 in esophageal carcinogenesis, we examined the expression of this enzyme in normal squamous epithelium (n = 42), squamous dysplasia [high-grade dysplasia (HGD, n = 41; low-grade dysplasia (LGD, n = 33)]; carcinoma in situ (n = 16), mucosal invasive carcinoma (n = 18), and advanced SCC (n = 45). Immunohistochemistry showed a significantly high COX-2 expression in HGD compared with other lesions. The COX-2 score, an index determined by intensity and positivity of COX-2 staining (maximum 3.0), was 0.29 +/- 0.04 in normal esophagus, 1.75 +/- 0.11 in LGD, 2.89 +/- 0.05 in HGD, 2.17 +/-0.18 in CIS, 1.95 +/- 0.22 in mucosal invasive carcinoma, and 1.81 +/- 0.08 in advanced SCC. Results of reverse transcription-PCR assays confirmed those obtained by immunohistochemistry. COX-2 expression correlated with proliferation activity assessed by the proliferating cell nuclear antigen index in dysplastic lesions (P = 0.001) but not in SCCs. COX-2 expression in SCC did not correlate with various clinicopathological parameters including prognosis. Our results indicate that COX-2 is a sensitive marker for HGD and suggest that COX-2 may be involved in early stages of squamous carcinogenesis of the esophagus.

Mammaglobin B gene as a novel marker for lymph node micrometastasis in patients with abdominal cancers.

Mammaglobin B is a recently-isolated gene speculated to belong to the uteroglobin gene family and is overexpressed in primary breast cancers. We investigated mammaglobin B mRNA expression in various cancers of the digestive system. Given the absence of mammaglobin B expression in normal lymph nodes, we also assessed the usefulness of mammaglobin B as a marker for lymph node micrometastases in cancer patients. Mammaglobin B gene transcripts were frequently detected by reverse transcriptase-polymerase chain reaction (RT-PCR) assay in primary tumors of the esophagus (2/3), stomach (7/7), colon (15/15), pancreas (4/6), common bile duct (6/6), cholangioma (2/2) and gall bladder (1/1). Mammaglobin B overexpression was observed in three of 15 cases (20%) of colon cancer, suggesting its possible contribution to colon carcinogenesis. Down-regulated mammaglobin B expression was observed in hepatoma cells in comparison with corresponding non-cancerous livers (3/3). RT-PCR assay of mammaglobin B detected 14 of 15 histologically positive lymph nodes from patients with gastric cancer, colon cancer and cholangioma. Seven of 32 (22%), three of nine (33%), and three of seven (43%) histologically negative nodes from patients with gastric, colon and cholangiocellular carcinoma, respectively, were found to express mammaglobin B mRNA. Our results showed that expression of mammaglobin B was frequently detected in cancers originating in digestive organs, especially adenocarcinomas, and that mammaglobin B gene detected by RT-PCR may be a potentially useful molecular marker for lymph node micrometastases of various digestive organ cancers.

Overexpression of cyclooxygenase-2 in carcinoma of the pancreas.

The level of cyclooxygenase (COX)-2 has been investigated recently in various human carcinomas. In the present study, we examined the distribution and extent of COX-2 protein in human pancreatic tumors using immunohistochemistry. A strong expression of COX-2 protein was present in 23 of 52 (44%) pancreatic carcinomas, a moderate expression was present in 24 of 52 (46%) pancreatic carcinomas, and a weak expression was present in 5 of 52 (10%) pancreatic carcinomas. In contrast, benign tumors showed weak expression or no expression of COX-2, and only islet cells displayed COX-2 expression in normal pancreatic tissues. Overexpression of COX-2 in carcinoma tissues was also confirmed by Western blot analysis. Furthermore, consistent with the results at protein levels, reverse transcription-PCR analyses indicated that COX-2 mRNA was overexpressed in 7 of 13 (54%) carcinomas, but in none of 3 benign tumors. Our findings suggest that COX-2 inhibitors might be potentially effective against pancreatic carcinomas and that COX-2 may be involved in certain biological processes in pancreatic islets.

Increased expression of COX-2 in nontumor liver tissue is associated with shorter disease-free survival in patients with hepatocellular carcinoma.

Recent studies have shown increased levels of cyclooxygenase-2 (COX-2) in a variety of human malignancies including hepatocellular carcinoma (HCC), but little is known about the prognostic value of COX-2 in HCC or its associated nontumor liver tissue. We examined the expression of COX-2 protein by immunohistochemistry in 53 patients with HCCs whose corresponding nontumor tissues were hepatitis C virus-related chronic hepatitis (n = 21) and cirrhosis (n = 32). Samples of nine histologically normal livers and eight precancerous dysplasias were also analyzed. The level of COX-2 increased from normal liver to chronic hepatitis to cirrhosis. The majority of cirrhotic livers (81%) displayed marked COX-2 expression. In dysplasias, COX-2 expression was mainly moderate or strong (88%). In HCC, 17% of samples displayed a high COX-2 expression, and 37% of samples expressed COX-2 at a moderate level. Concordant results were obtained with reverse transcription-PCR and Western blot analyses. Clinicopathological survey indicated a significant correlation between COX-2 expression and differentiated carcinoma (P = 0.019). Although there was no correlation between COX-2 expression in HCC and prognosis, a striking difference was found between COX-2 expression in nontumor tissue and shorter disease-free survival (P = 0.0132). Moreover, high COX-2 expression in nontumor tissue was significantly correlated with the presence of active inflammation (P < 0.0001). The present findings suggest that COX-2 expression in nontumor tissue may play a positive role in relapse of HCC after surgery.