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Background We investigated the role of A2B-adenosine receptor (A2BAR) in regulating immunosuppressive metabolic stress in the tumor microenvironment (TME). Novel A2BAR antagonist PBF-1129 was tested for anti-tumor activity in animals and evaluated for safety and immunological efficacy in a phase-I clinical trial in non-small-cell lung cancer (NSCLC) patients. Methods Anti-tumor efficacy of A2BAR antagonists and impact on metabolic and immune TME was evaluated in lung, melanoma, colon, breast and EGFR-inducible transgenic cancer models. Employing Electron Paramagnetic Resonance, we assessed changes in TME metabolic parameters including pO2, pH, and inorganic phosphate (Pi) during tumor growth and evaluated the immunological effects of PBF-1129, including its pharmacokinetics, safety, and toxicity in NSCLC patients. Results Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial Pi emerged as a correlative and cumulative measure of TME stress and immunosuppression. A2BAR inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ecto-nucleotidases, increased expression of adenosine deaminase (ADA), decreased tumor growth and metastasis, increased IFN-γ-production and enhanced the efficacy of anti-tumor therapies following combination regimens in animal models (anti-PD-1 vs. anti-PD-1 plus PBF-1129 treatment hazard ratio [HR] = 11.74, 95% CI = 3.35 to 41.13, n = 10, P Conclusions Data identify A2BAR as a valuable therapeutic target to modify metabolic and immune TME to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

Supplementary Figure 1 Legend from Inhibition of Epidermal Growth Factor Receptor Signaling Elevates 15-Hydroxyprostaglandin Dehydrogenase in Non–Small-Cell Lung Cancer

Supplementary Table 1 and Figure 1 from Inhibition of Epidermal Growth Factor Receptor Signaling Elevates 15-Hydroxyprostaglandin Dehydrogenase in Non–Small-Cell Lung Cancer

We herein report a rare case of intraductal papillary mucinous neoplasm with a pancreatogastric fistula in an elderly Japanese man admitted to our hospital. The pancreatogastric fistula was confirmed using endoscopic retrograde pancreatography via a cannulated guidewire placed in the stomach. Six months after admission, the patient was diagnosed with intraductal papillary mucinous carcinoma. A pancreatogastric fistula is generally a rare complication of intraductal papillary mucinous neoplasm. It was caused by mechanical penetration in this case. Interestingly, we also observed endoscopic and histochemical mucosal changes in the fistula.

IgG4-related autoimmune hepatitis (IgG4-AIH) is characterized by hepatic inflammation and is considered an IgG4-related disease. Several inflammatory pseudotumors (IPTs) are also considered as IgG4-related diseases;however, there have been no reports of cases wherein both diseases occurred concurrently. An older adult with liver dysfunction was admitted to the hospital and was diagnosed with IgG4-AIH following a liver biopsy;IgG4-positive plasma cell infiltration in the portal tract and high serum IgG4 concentration were detected. A few months following biopsy, imaging studies revealed two IPTs in the liver. The patient was diagnosed with cryptogenic organized pneumonia several months after imaging and was treated with steroids in a different hospital. Her liver dysfunction improved, and one of the two IPTs disappeared in response to steroid treatment. The following is an account of a rare case of IgG4-AIH with IPTs of the liver.

We report a rare case of undifferentiated-type intramucosal gastric cancer that occurred in the fornix of the stomach without Helicobacter pylori infection, which consisted mainly of poorly differentiated adenocarcinoma. A 49-year-old man visited our hospital for a follow-up endoscopic examination of a small depressed lesion of the gastric fornix detected by surveillance esophagogastroduodenoscopy. On magnifying endoscopy with blue laser imaging, the depressed lesion (approximately 10 mm in diameter) was regarded as undifferentiated-type early gastric cancer that proved to be a poorly differentiated adenocarcinoma by histological examination of biopsied specimens. The cancerous lesion was successfully treated with endoscopic submucosal dissection and microscopically showed an intramucosal cancer that invaded the whole mucosal layer with predominant growth of a poorly differentiated adenocarcinoma component. The patient status was verified as Helicobacter pylori-naive according to the strict diagnostic criteria, thereby confirming this case as an undifferentiated-type Helicobacter pylori-uninfected gastric cancer. Helicobacter pylori-uninfected intramucosal poorly differentiated adenocarcinoma occurring in the gastric fornix has not been previously reported.

Paraganglioma and pheochromocytoma are rare neuroendocrine neoplasms that originate from chromaffin cells. In many of these tumors, several mutations are reported to occur in the genes of germline and/or somatic cells. A case of paraganglioma in the posterior mediastinum with highly malignant potential is reported. The patient had a rapid clinical course, and it was difficult to reach the final diagnosis. The initial diagnosis on fine-needle aspiration biopsy was a gastrointestinal stromal tumor (GIST) arising from the esophagus. Although radiation therapy was effective for the main tumor, the lung metastases did not respond sufficiently to several tyrosine kinase inhibitors. Autopsy and immunohistochemical examination using a battery of different markers resulted in a final diagnosis of malignant paraganglioma. Next-generation sequencing revealed several gene mutations and copy number variations, including of fumarate hydratase (FH), neurofibromatosis type-1 (NF1) and RET. Those gene alterations may contribute to the pathogenesis of this malignant phenotype to a certain extent. To confirm this, further cases and studies are required. In addition, it should be noted that histological examination of a small piece of tumor might have sampling bias and could cause misdiagnosis.

We herein report the case of a 78-year-old woman with an intraductal tumor with scant mucin production in a moderately dilated main pancreatic duct that resembled an intraductal tubulopapillary neoplasm (ITPN) on imaging. An endoscopic transpapillary forceps biopsy enabled an accurate preoperative diagnosis of the tumor as an oncocytic type intraductal papillary mucinous neoplasm (IPMN) of the pancreas microscopically showing papillary growth consisting of oncocytic cells with a typical mucin expression profile, although with few intraepithelial lumina containing mucin. This is the first case of an oncocytic type IPMN mimicking an ITPN that was able to be diagnosed preoperatively.

Background/aims A multifunctional snare SOUTEN has a sharp tip at the top of the snare loop that enables incision of the mucosa, dissection of the submucosal layer, and snaring of lesion. This study assessed the efficacy and safety of complete endoscopic resection of colorectal neoplasia using SOUTEN. Methods We analyzed the rates of gross en bloc resection and complete resections of 108 consecutive tumors from 69 patients resected by precutting endoscopic mucosal resection (precutting), hybrid endoscopic submucosal dissection (hybrid), or conventional endoscopic submucosal dissection (conventional) using SOUTEN. Results Out of the 108 tumors, 50 were resected by precutting, 27 were resected by hybrid after attempting precutting, and the remaining 31 were resected by conventional after attempting precutting and hybrid resections. The median tumor sizes were 14.5 mm for precutting, 16.4 mm for hybrid, and 21.1 mm for conventional. The success rate of gross en bloc resection and histological complete resection were 100% and 94.0% for precutting, 96.4% and 96.4% for hybrid, and 100% and 100% for conventional method, respectively. No procedure-related complication occurred. Conclusion By using SOUTEN, precutting and hybrid were successfully performed on 10-30 mm tumors with a shorter procedure time than conventional without major complications.

Background Percutaneous endoscopic gastrostomy (PEG) is the most common method of enteral nutrition in patients who require long-term tube feeding. According to meta-analyses, administration of systemic prophylactic antibiotics for PEG reduces peristomal infection. However, with several recent developments in the procedure and instruments, the risk of infection might have been reduced. The aim of this study was to evaluate the use of systemic antibiotic prophylaxis for a modified introducer method of PEG. Methods This prospective, randomized, double-blind trial assessed 278 patients undergoing PEG for inclusion. Ninety-one patients with an indication for PEG who gave informed consent to participate were randomized. Forty-six patients received prophylactic ampicillin and 45 patients received a placebo. A modified introducer method of PEG using a Seldinger PEG kit was performed. The primary outcome was the occurrence of clinically evident wound infection within 3 days after PEG. Results Wound infection within 3 days was observed in none in the prophylaxis group and in 1 patient in the control group (P=0.4945). There was no significant difference between 2 groups in the other parameters, including peristomal infection within 7 days, overall infection, white blood cell counts, C-reactive protein level, and successive rate of finishing antibiotics. Conclusions For wound infection within 3 days, noninferiority of the placebo group to the antibiotics group was preliminarily suggested with our criteria, but not for peristomal infection within 7 days. More strict criteria for noninferiority should be examined in a further large sample study.

The incidence of esophageal adenocarcinoma (EAC) in cases with long-segment Barrett's esophagus (BE) has not been investigated in Japan. The aim of this study is to investigate the incidence of EAC in Japanese cases with long-segment BE prospectively.This is a multicenter prospective cohort study investigating the incidence rate of EAC in patients with BE with a length of at least 3 cm. Study subjects received index esophagogastroduodenoscopy at the time of enrollment, and they were instructed to undergo yearly follow-up esophagogastroduodenoscopy. Patients in whom EAC was diagnosed in the endoscopic examinations underwent subsequent treatment, and their prognosis was observed.Of 215 enrolled patients, six (2.8%) were initially diagnosed with EAC at the enrollment. Among the remaining 209 patients, 132 received at least one follow-up esophagogastroduodenoscopy. In this follow-up, three EACs developed in 251 observed patient-years (incidence rate: 1.2% per year). Most of the EACs detected at the initial endoscopic examination (5/6, 83%) were already at advanced stages. Meanwhile, all the three lesions detected in the follow-up esophagogastroduodenoscopies were identified as early cancers and subjected to curative resection.The incidence rate of EAC in Japanese cases with long-segment BE was calculated to be 1.2% in a year.

Anaplastic carcinoma is a rare pancreatic cancer, and the malignant transformation of a heterotopic pancreas is also rare. We herein report a case of an elderly woman with a mass of unknown origin in the abdominal cavity. Computed tomography identified the extent of the tumor but not the organ of origin. The abdominal tumor eventually metastasized to the liver and lung. An autopsy and immunohistochemical examination revealed an anaplastic carcinoma possibly originating in an ectopic pancreas.

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin–ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected.

To clarify the role of DNA methylation in the silencing of the expression of cyclin-dependent kinase inhibitor p57KIP2 seen in certain tumors, we investigated the methylation status of its 5′ CpG island in various tumor cell lines and primary cancers. Dense methylation of the region around the transcription start site was detected in 1 out of 10 colorectal, 2 out of 8 gastric, and 6 out of 14 hematopoietic tumor cell lines and in 5 out of 35 (14%) gastric, 6 out of 20 (30%) hepatocellular, and 2 out of 18 (11%) pancreatic cancers; 7 out of 25 (28%) acute myeloid leukemia cases also showed methylation of the p57KIP2 gene, which strongly correlated with the CpG island methylator phenotype (P

When DNA damage is induced by unprogrammed extrinsic events, activating-cell-cycle checkpoints delay cell-cycle progression in the G1 or G2 phases and allow repair of a damaged template. In this study, we evaluated changes in gene expression upon radiation-induced G2 cell-cycle arrest using Chinese hamster ovary (CHO) cells. T-fimbrin, an actin-binding protein, was overexpressed in CHO cells in which G2 arrest had been induced by X-radiation. Northern blot analysis revealed that T-fimbrin gene expression was induced not only by X-radiation but also by a topoisomerase II inhibitor, etoposide. Transfection of CHO cells with a vector encoding T-fimbrin antisense RNA demonstrated that reduced T-fimbrin expression induced alterations in cell-cycle control; radiation-induced G2 arrest was short and decreased in cells transfected with antisense T-fimbrin. Additionally, T-fimbrin gene expression was suppressed in a human colorectal cancer cell line, SW948, because of promoter-specific DNA methylation. These results suggest that downregulation of T-fimbrin may be involved in cancer development through G2/M cell-cycle control in mammalian cells.

Cyclooxygenase 2 plays a critical role in the development of gastrointestinal cancers in both human and animal models. About 80% of the gastric cancer showed a high level of expression of cyclooxygenase 2, but a subset of cases do not express without unknown reason. Aberrant methylation of CpG island of COX-2 was examined by using a series of gastric cancer cell lines and primary gastric cancers. Two out of 8 cell lines (25%) and 11 out of 93 (12%) primary cancers showed aberrant methylation of the 5' region of COX-2. Methylation of COX-2 was closely associated with loss of expression and treatment of methylation inhibitor, 5-deoxy-2'-azacytidine restored the expression of COX-2. A combined treatment of 5-deoxy-2'-azacytidine and a histone deacetylese inhibitor, trichostatin A, restored re-expression of the gene synergistically and chromatin immunoprecipitation analysis revealed that histone of methylated COX-2 promoter is deacetylated, indicating the role of cytosine methylation and histone deacetylation in the silencing of the gene. These results indicate that a subset of gastric cancer with COX-2 methylation evolves through the pathway that is independent of COX-2 expression and that COX-2 inhibitor may not be useful to induce apoptosis in these cases.

The 14-3-3 sigma gene has been implicated in G2/M cell cycle arrest by p53. Frequent inactivation of the 14-3-3 sigma gene by hypermethylation of CpG islands has recently been reported in human breast carcinoma. The aim of this study was to examine the methylation status of CpG islands of the 14-3-3 sigma gene in hepatocellular carcinoma (HCC). The methylation status of the 14-3-3 sigma gene was evaluated in four normal liver tissues and 19 paired specimens of carcinoma and adjacent non-tumorous liver tissues using bisulfite-single strand conformation polymorphism (bisulfite-SSCP), a combination of sodium bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. The 14-3-3 sigma protein expression was examined by immunohistochemical staining. Hypermethylation of CpG islands of the 14-3-3 sigma gene was detected in 89% (17/19) of the HCC tissues but not in any of the four normal liver tissues. All of the 14 methylation-positive HCC samples analysed by immunohistochemistry showed loss of 14-3-3 sigma expression, while both of the methylation-negative HCC samples retained the expression, and a significant correlation was found between methylation and loss of expression. Lower levels of methylation were detected in adjacent non-tumorous liver tissues (6/16 in cirrhotic tissues and 1/3 in chronic hepatitis tissues), but the 14-3-3 sigma expression was retained in all of these tissues. In a methylation-positive HCC cell line, HLE, 5-aza-2'-deoxycytidine (5-aza-dC)-induced demethylation of CpG islands led to reactivation of gene expression, indicating that hypermethylation plays a causal role in inactivation of the 14-3-3 sigma gene in HCC. Hypermethylation and the resulting loss of expression of the 14-3-3 sigma gene corresponds to one of the most common abnormalities reported to date in HCC, suggesting their crucial role in the development and/or progression of HCC.

Protein signals obtained directly from frozen lung tissue sections using MALDI-MS were used to predict nodal involvement and survival in resected non-small cell lung cancer (NSCLC). We have identified a list of these protein signals and further evaluated their prognostic values for NSCLC using immunohistochemistry (IHC). Kaplan-Meier analysis was used to assess the mortality risk associated with the prognostic protein IHC-staining intensities. The combined IHC scores of calmodulin, thymosin β4, and thymosin β10 were found to be correlated with NSCLC patient survival (p = 0.004). Furthermore, low cofilin-1 IHC-staining intensity was found to be correlated with a better outcome for patients with negative lymph node status (p = 0.006) while high cofilin-1 IHC-staining intensity was found to be correlated with a better outcome for patients with positive node status (p = 0.034). In conclusion, the prognostic protein signals selected using MALDI-MS can be identified and tested by IHC in formalin-fixed tissue samples. MALDI-MS-derived protein signals can be potentially translated to a conventional clinical setting to aid in the prognosis of patients with NSCLC at the molecular level.

Recent technological developments in proteomic analysis are bringing us new insights into the molecular classification of tumours. Although proteomic analysis in cancer profiling is still under development both in terms of the instruments used and the data analytical tools, this method has great potential advantages for the analysis of biospecimens of many types. Direct measurement of abnormally expressed or modified proteins in the tumour tissue and/or patient blood may be an effective approach for discovering new biomarkers. Proteomics has the significant advantage of being able to discern not only changes in expression levels but also in post-translational modifications. Thus, the proteomics approach to protein profiling and biomarker discovery uncovers biomarkers from a different viewpoint than microarray analysis. This review summarizes the range of proteomics technologies employed for cancer profiling, and how they have been used to derive new classification models for human lung cancer.

Aberrant methylation of CpG islands can be a good molecular marker for identifying genes inactivated in cancer. We found the proapoptotic gene HRK to be a target for hypermethylation in human cancers and examined the role of such methylation in silencing the gene's expression.Methylation of HRK was evaluated by bisulfite-PCR and bisulfite sequencing in a group of colorectal and gastric cancer cell lines and primary cancers. Gene expression and histone acetylation were examined by reverse transcription-PCR and chromatin immunoprecipitation analyses, respectively. Apoptosis of cancer cells after treatment with a DNA methyltransferase inhibitor and/or histone deacetylase inhibitor was examined with fluorescence-activated cell-sorting analysis.The region around the HRK transcription start site was methylated in 36% of colorectal and 32% of gastric cancer cell lines and was closely associated with loss of expression in those cell types. HRK expression was restored by treatment with a methyltransferase inhibitor, 5-aza-deoxycytidine, and enhanced further by addition of histone deacetylase inhibitor trichostatin A or depsipeptide. Such restoration of HRK expression was well correlated with induction of apoptosis and enhancement of Adriamycin-induced apoptosis. Expression of other proapoptotic genes, including BAX, BAD, BID, and PUMA, was unaffected by treatment with 5-aza-deoxycytidine. Aberrant methylation of HRK was also frequently detected in primary colorectal cancers that showed methylation of multiple genes, including p16INK4A and hMLH1, and was associated with wild-type p53.HRK methylation can be a useful molecular target for cancer therapy in a subset of colorectal and gastric cancers.

Mitotic checkpoints prevent errors in chromosome segregation that can lead to neoplasia. Therefore, it is notable that gastric cancers often show impaired checkpoint function. In the present study, we examined the functional consequences of epigenetic inactivation of the mitotic checkpoint gene CHFR in gastric cancers. CHFR expression was silenced by DNA methylation of the 5' region of the gene in 20% of the gastric cancer cell lines tested and in 39% of primary gastric cancers; expression could be restored by treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor. In addition, histones H3 and H4 were found to be deacetylated in cell lines showing aberrant methylation, indicating a role for histone deacetylation in the methylation-dependent gene silencing. Cells not expressing CHFR showed impaired checkpoint function, which led to nuclear localization of cyclin B1 after treatment with docetaxel or paclitaxel, two microtubule inhibitors. Apparently, the absence of CHFR is associated with sensitivity of cells to mitotic stress caused by microtubule inhibition, and restoration of CHFR expression by 5-aza-2'-deoxycytidine or adenoviral gene transfer restored the checkpoint. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in gastric cancer. Moreover, the aberrant methylation of CHFR appears to be a good molecular marker with which to predict the sensitivity of gastric cancers to microtubule inhibitors.

We have been investigating gene expression profiles in non-small cell lung cancers (NSCLCs) to identify molecules involved in pulmonary carcinogenesis and select which genes or gene products might be useful as diagnostic markers or targets for new molecular therapies. Here we report evidence that the cytochrome c oxidase (CCO) assembly protein COX17 is a potential molecular target for treatment of lung cancers. By semiquantitative reverse transcription-PCR, we documented increased expression of COX17 in all of 8 primary NSCLCs and in 11 of 15 NSCLC cell lines examined, by comparison with normal lung tissue. Treatment of NSCLC cells with antisense S-oligonucleotides or vector-based small interfering RNAs of COX17 suppressed expression of COX17 and also the activity of CCO, and suppressed growth of the cancer cells. Because our data imply that up-regulation of COX17 function and increased CCO activity are frequent features of lung carcinogenesis, we suggest that selective suppression of components of the CCO complex might hold promise for development of a new strategy for treating lung cancers.

To date, no single or multiple molecular markers have been successful in predicting sensitivity of individual patients to anti-cancer drugs. As the nature of a specific cancer is considered to be defined by the proteins being expressed in the tumor cells, systematic analysis of gene-expression profiles may provide information reflecting sensitivity of a given tumor to certain drugs. Recent progress in genome technology has enabled us to examine expression profiles of thousands of genes in a single experiment. We used this approach to examine 13 xenografts of human tumors implanted into nude mice for sensitivity to an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), ZD1839 (Iressa). To identify genes that might be associated with sensitivity to this drug we used a cDNA microarray representing 23,040 genes to analyze expression profiles of the 13 xenografts and identified 114 genes whose expression levels correlated significantly with sensitivity of the tumors to ZD1839. We then investigated alteration of expression profiles in response to the ZD1839 treatment in four non-small cell lung cancer (NSCLC) xenografts, of which two (LC6 and LC11) were sensitive and the other two (Lu116 and L27) were resistant to this EGFR-TKI. Systematic analysis of expression at various time points during oral treatment for 14 days, compared with corresponding untreated samples, identified a set of genes whose expression levels changed in the two sensitive tumors but not in the two resistant tumors. The data obtained here should provide useful information on the molecular mechanism underlying clinical responses to EGFR-TKIs, aid the development of novel therapies for lung cancer, and potentially identify predictive molecular markers for sensitivity to ZD1839.

To investigate genes involved in pulmonary carcinogenesis and those related to sensitivity of nonsmall cell lung cancers (NSCLCs) to therapeutic drugs, we performed cDNA microarray analysis of 37 NSCLCs after laser-capture microdissection of cancer cells from primary tumors. A clustering algorithm applied to the expression data easily distinguished two major histological types of non-small cell lung cancer, adenocarcinoma and squamous cell carcinoma. Subsequent analysis of the 18 adenocarcinomas identified 40 genes whose expression levels could separate cases with lymph-node metastasis from those without metastasis. In addition, we compared the expression data with measurements of the sensitivity of surgically dissected NSCLC specimens to six anti-cancer drugs (docetaxel, paclitaxel, irinotecan, cisplatin, gemcitabine, and vinorelbine), as measured by the CD-DST (collagen gel droplet embedded culture-drug sensitivity test) method. We found significant associations between expression levels of dozens of genes and chemosensitivity of NSCLCs. Our results provide valuable information for eventually identifying predictive markers and novel therapeutic target molecules for this type of cancer.

Recent expression profile analyses revealed that lung adenocarcinomas can be divided into several subgroups with diverse pathological features. Because cellular heterogeneity of tumors can confound these analyses, we used laser capture microdissection and microarray expression analysis to characterize the molecular profiles of lung adenocarcinomas. We found 45 genes delineating smokers and nonsmokers that were located at chromosomal loci frequently altered in non-small cell lung cancers, and 27 genes, which were differentially expressed between survivors and nonsurvivors 5 years after surgery. These results are consistent with the hypothesis that the abnormal expression of genes involved in maintaining the mitotic spindle checkpoint and genomic stability, e.g., hBUB3, hZW10, and APC2, contribute to the molecular pathogenesis and tumor progression of tobacco smoke-induced adenocarcinoma of the lung.

Aberrant 5' CpG island methylation is an alternative mechanism of gene inactivation during the development of cancer as demonstrated for several tumor-suppressor genes. Also, marked relationship of microsatellite instability (MSI) and DNA methylation has been reported in sporadic colorectal cancer, which is a result of epigenetic inactivation of hMLH1 in association of promoter hypermethylation. In the present study, we investigated the 5' CpG island hypermethylation of hMLH1, E-cadherin and p16 in 61 primary gastric cancers (GCs) by using combined bisulfite restriction analysis (COBRA) and methylation-specific PCR (MSP), and their MSI status. Of 61 GCs investigated, 5 (8.1%) tumors presented hMLH1 methylation, 16 (26.2%) and 25 (40.9%) showed E-cadherin and p16 methylation respectively, and 8 (13.1%) presented high-frequency MSI (MSI-H). Of the 8 MSI-H patients, 5 presented hMLH1 methylation, whereas no low-frequency MSI (MSI-L) and microsatellite stable (MSS) cases exhibited hMLH1 methylation (5/8 vs. 0/43, p < 0.00001). Furthermore, these patients also presented E-cadherin and p16 hypermethylation. Our data showed a significant correlation between hMLH1 methylation and MSI in GC, and suggested that a common mechanism of aberrant de novo methylation can be postulated in these cancers.

A biomarker signature to identify individuals at-risk for lung cancer would be of great clinical value. We hypothesized that the bronchial epithelium expresses a subset of proteins that once increased confer an elevated risk for lung cancer. We have therefore developed a proteomic approach to assess the risk for lung cancer. From our previous studies, we selected 11 matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) features to distinguish normal bronchial epithelium and low grade preinvasive lesions from high grade preinvasive lesions and invasive lung tumors. We tested this signature in an independent validation set consisting of bronchial specimens from 60 patients. The prediction accuracy was 78% with 73% sensitivity and 81% specificity. There was a 19-fold increased risk of having lung cancer for individuals with a signature intensity above the third quartile (95% CI, 5.65-64.81). We identified 10 of the 11 features corresponding to 7 proteins, thymosin ß4, ubiquitin, acyl-coA binding protein, cystatin A, S100A11, cytochrome c and macrophage migration inhibitory factor. The identity of these proteins was validated by immunohistochemistry, Western blotting and MALDI imaging mass spectrometry (IMS). MALDI IMS localized the candidate biomarker proteins specifically to high grade preinvasive lesions and invasive tumors. This novel proteomic signature may facilitate the selection and monitoring of high risk individuals for lung cancer early detection and chemoprevention strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2727.

Distant metastasis is one of the crucial parameters determining the type of treatment and prognosis of patients. Previous studies discovered important factors involved in multiple steps of metastasis, the precise mechanisms of metastasis still remain to be clarified. To identify genes associated with this complicated biological feature of cancer, we analyzed expression profiles of 16 metastatic brain tumors derived from primary lung adenocarcinoma (ADC) using cDNA microarray representing 23,040 genes. We applied bioinformatic algorithm to compare the expression data of these 16 brain metastatic loci with those of 37 primary NSCLCs including 22 ADCs, and found that metastatic tumor cells has very different characteristics of gene expression patterns from primary ones. Two hundred and forty-four genes that showed significantly different expression levels between the two groups included plasma membrane bounding proteins, cellular antigens, and cytoskeletal proteins that might play important roles in altering cell-cell communication, attachment, and cell motility, and enhance the metastatic ability of cancer cells. Our results provide valuable information for development of predictive markers as well as novel therapeutic target molecules for metastatic brain tumor of ADC of the lung.