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A 32-year-old Japanese man presented to our hospital with a 5-month history of painful nodules on the fingers. He had previously developed left atrial myxoma followed by cerebral embolism and had undergone resection of the cardiac tumor 4 months before his first visit. His family history was unremarkable. Physical examination showed subcutaneous nodules 4 to 17 mm in size on all finger pads (Figure 1A and 1B). The tumors were smooth and as hard as cartilage. Hand radiographs showed irregularly shaped subcutaneous calcification corresponding to the nodules (Figure 2), and postoperative examination of the 3-dimensional computed tomographic reconstruction indicated the tumors more clearly (Figure 3). Physical examination and diagnostic imaging detected no tumors in other sites, including the lower extremities. The blood test results, including endocrine examination, were unremarkable. Figure 1. Subcutaneous nodules on all finger pads: the left …

Interferon-induced transmembrane protein (IFITM) is reported to be frequently overexpressed in colorectal tumors. This study aimed to determine the usefulness of detecting fecal IFITM messenger RNA (mRNA) by real-time reverse-transcription polymerase chain reaction (RT-PCR) for colorectal cancer (CRC) screening. This pilot study included 21 patients with CRC and 23 healthy controls. Total RNA was isolated from the feces of the patients, and the expression levels of the mRNA of IFITM1, IFITM2 and IFITM3 were measured by real-time RT-PCR to detect CRC. Receiver operating characteristic curves of respective genes were generated, and the area under the curve (AUC), sensitivity and specificity were determined. When the 44 patients were analyzed, the AUCs of fecal IFITM1, IFITM2 and IFITM3 expression analysis were 0.82, 0.80 and 0.65, respectively. The sensitivities were 67% [14/21; 95% confidence interval (CI) 43–85%], 67% (14/21; 95% CI 43–85%) and 71% (15/21; 95% CI 48–89%), respectively; and the specificities were 96% (1/23; 95% CI 78–100%), 96% (1/23; 95% CI 78–100%) and 61% (9/23; 95% CI 39–80%), respectively. When IFITM1 and IFITM2 were combined, the sensitivity was 86% (18/21; 95% CI 64–97%) and the specificity was 96% (1/23; 95% CI 78–100%). The fecal expression analysis of IFITM1 and IFITM2 mRNA by real-time RT-PCR for CRC screening exhibited high specificities, and the sensitivity was further improved by combining IFITM1 and IFITM2.

Morphologically, early colorectal tumors are divided into two groups, protruded-type tumors and flat-type tumors. Although some studies have shown genetic alterations in protruded-type tumors, little is known about genetic and epigenetic alterations in flat-type tumors, as well as pT1 (early invasive) colorectal cancers (CRCs). In the current study, we compared the frequencies of genetic and epigenetic alterations of the RAS-RAF and Wnt signaling pathways in flat-type and protruded-type tumors. In addition, we investigated the relationship between those alterations and invasive potential of pT1 CRCs. Methylations of RASSF2, O-6-methylguanine-DNA methyltransferase (MGMT), Wnt inhibitory factor-1 (WIF-1), EPHB2, CDKN2A and MLH1 were detected in 44.3, 30.3, 81.4, 7.5, 43.6 and 13.4% of the 307 early colorectal tumors, respectively. Mutations of KRAS, BRAF, catalytic subunit alpha of phosphatidylinositol 3'-kinase (PIK3CA) and beta-catenin were detected in 25.4, 4.6, 1.6 and 9.4% of those tumors, respectively. Methylations of MGMT, WIF-1 and CDKN2A were detected in significantly higher percentages of protruded-type tumors than in flat-type tumors. Mutation of at least one gene was detected in a significantly higher percentage of flat-type tumors than in protruded-type tumors. RASSF2 methylation was correlated significantly with KRAS, BRAF or PIK3CA mutation. Multiple logistic analysis showed that lymphatic invasion and RASSF2 methylation with KRAS, BRAF or PIK3CA mutation were independent risk factors for venous invasion in pT1 CRCs. In conclusion, since genetic alterations of these pathways have frequently occurred in flat-type tumors, flat-type tumors seem to have a distinct genetic profile different from that of protruded-type tumors. RASSF2 methylation with oncogenic activation is a promising biomarker for predicting invasive potential of pT1 CRCs.

Morphologically, colorectal adenomas can be divided into two groups, protruded-type and flat-type. However, the accurate frequencies of genetic and epigenetic alterations in flat-type colorectal advanced adenomas (laterally spreading tumors) have remained largely unknown. In the current study, we investigated genetic and epigenetic alterations in 101 flat-type colorectal advanced adenomas and 68 protruded-type colorectal advanced adenomas by using direct DNA sequencing and quantitative real-time PCR (MethyLight), respectively. KRAS mutation was detected in a significantly higher percentage of flat-type adenomas (35%) than in protruded-type adenomas (13%). When the samples were limited to the tumors in the distal colon, the difference of KRAS mutation was still significant. KRAS mutation in G-to-A transitions at codons 12 and 13 was detected in a significantly higher percentage of flat-type adenomas (26%) than in protruded-type adenomas (9%). BRAF and beta-catenin mutations were detected in 3 and 8% of the 101 flat-type adenomas, respectively. No significant difference was found between frequencies of those mutations in flat-type adenomas and protruded-type adenomas. Methylations of MGMT, CDKN2A (p16) and MLH1 were detected in 28, 33 and 9% of the 101 flat-type adenomas, respectively. CDKN2A methylation was detected in a significantly lower percentage of flat-type adenomas than in protruded-type adenomas (63%). Methylation of at least one gene was detected in a significantly lower percentage of flat-type adenomas (54%) than in protruded-type adenomas (78%). In conclusion, KRAS mutation was frequently detected in flat-type advanced adenomas and the mutational patterns in most of them with KRAS mutations were a transition from G-to-A. Therefore, these genetic alterations seem to play an important role in the development of flat-type advanced adenomas, especially in the distal colon. Epigenetic alterations infrequently occurred in flat-type advanced adenomas, suggesting that they have different genetic and epigenetic alterations from those of protruded-type advanced adenomas.

Pressure dependence of specific heat of CeRh 2 Si 2 has been measured by a thermal relaxation method above 11 K. Specific heat jump arising from the antiferromagnetic transition is observed at 36 K and 0 GPa. With increasing pressure, the anomaly is suppressed and shifted to lower temperatures. The pressure dependence of the specific heat anomaly is consistent with the results of electric resistance measured simultaneously.

Gastric cancers with and those without high-frequency microsatellite instability (MSI-H) represent distinctive pathways of carcinogenesis. The aim of this study was to clarify if expression of p53 related genes involved in angiogenesis is differentially regulated between these cancers. We systematically analyzed the expression of vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2), thrombospondin 1 (THBS1), and brain-specific angiogenesis inhibitor 1 (BAI1), and we correlated the results with microvessel count (MVC), MSI status, p53 mutations, and prostaglandin-endoperoxide synthase 2 (PTGS2) expression in gastric cancers. Expression of VEGFA in carcinoma cells was immunohistochemically seen in 46% of 200 cases. VEGFA positivity was significantly associated with higher MVC, vascular invasion, lymph node and distant metastasis, and advanced tumor stage. FGF2 positivity was significantly associated with poor differentiation, depth of invasion, and higher MVC. VEGFA and FGF2 positivities and MVC were lower in MSI-H cancers than in MSI-L or MSS cancers. VEGFA expression was associated with both p53 mutations and PTGS2 expression. Methylation of the THBS1 gene was detected in 6 of 11 cancer cell lines and in 44% of 200 cases. THBS1 methylation was significantly associated with distal location, vascular invasion, distant metastasis, MSI-H, wild-type p53, and higher MVC. The prognosis was worst in patients with cancers that were VEGFA-positive and THBS1 methylation-positive. Gastric cancers with MSI-H were characterized by lower MVC, low frequency of VEGFA, FGF2, and PTGS2 overexpression, and high frequency of THBS1 methylation. Our results suggest that gastric cancers with and those without MSI-H represent distinctive pathways of carcinogenesis, including aberrant expression of factors regulating angiogenesis. The difference may be associated with less aggressive phenotype of these cancers with MSI-H and affect future molecular targeted therapeutics.

Aberrant activation and upregulation of the Wnt pathway is a key feature of many cancers. Wnt antagonists have recently attracted wide attention. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind to Wnt proteins directly and inhibit Wnt signaling pathway. It has been reported that WIF-1 expression is down regulated in several solid tumors and that WIF-1 is silenced by promoter hypermethylation in lung and colorectal cancer. By using RT-PCR, bisulfite sequence analysis, and methylation-specific PCR, we analysed expression and methylation of WIF-1 in cancer cell lines and freshly resected cancer tissues of the esophagus, stomach, colorectum, and pancreas. Downregulation of WIF-1 mRNA expression was observed in 61 (91.0%) of 67 cancer cell lines, 16 (80.0%) of 20 esophageal, 23 (74.2%) of 31 gastric, 41 (82.0%) of 50 colorectal, and six (75.0%) of eight pancreatic cancer tissues. Downregulation of WIF-1 expression was also observed at protein level. No significant association between WIF-1 downregulation and clinicopathological characteristics was found, suggesting that downregulation of WIF-1 expression is an early event in carcinogenesis of these cancers. Indeed, downregulation of WIF-1 expression was observed in 32 (72.7%) of 44 colorectal adenoma tissues and 18 (78.2%) of 23 early mucosal or submucosal colorectal carcinoma tissues. CpG island hypermethylation in the WIF-1 promoter region correlated with downregulation of WIF-1 expression in cancer cell lines and tissues. Treatment with demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), restored WIF-1 expression in cancer cell lines. A combined treatment of 5-aza-dC and a histone deacetylase inhibitor, trichostatinA, restored WIF-1 expression synergistically, indicating the role of cytosine methylation and histone deacetylation in the silencing of the WIF-1 gene. Transfection of the WIF-1 gene construct into TE-1 esophageal cancer cell lines or SW48 colon cancer cell lines lacking WIF-1 expression resulted in a significant inhibition on colony formation, cell proliferation, anchorage-independent growth in soft agar. TOPflash assay showed WIF-1 inhibits Wnt canonical signaling in these cell lines. These results suggest tumor suppressive function of WIF-1, due to its ability to inhibit Wnt signaling. Our results suggest that WIF-1 silencing due to promoter hypermethylation is an important mechanism underlying aberrant activation of the Wnt signaling pathway in carcinogenesis of the digestive organs. Modulation of the Wnt pathway, through reversal of WIF-1 silencing by demethylating agents, is a potential target for treatment and/or prevention of gastrointestinal cancers.

Expression of matrilysin-2, matrix metalloproteinase (MMP)-26, has been implicated in the progression of several types of human cancer. Matrilysin-2 has been reported to be a physiological and pathological activator of pro-MMP-9. The aim of this study was to examine matrilysin-2 expression and determine whether it is correlated with progression of human esophageal squamous cell carcinoma (ESCC). Semi-quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical analysis, zymography and an in vitro invasion assay were performed. Matrilysin-2 mRNA expression was undetectable or only faintly detected in non-tumor tissues, but its overexpression was detected in 24 of the 50 ESCC tissues. Matrilysin-2 overexpression was significantly correlated with depth of invasion, lymph node metastasis and an advance in pathological tumor node metastasis (pTNM) stage. Sections with immunostaining signals in >10% of carcinoma cells at the invasive front, which were observed in 46 of 100 cases, were judged to be positive for matrilysin-2 expression. Matrilysin-2 expression was significantly correlated with depth of invasion, lymph node and distant metastasis, advance in pTNM stage and recurrence. Expression of matrilysin-2 was significantly correlated with nuclear beta-catenin expression and MMP-9 expression. Patients with matrilysin-2-positive cancer had significantly shorter overall and disease-free survival periods than did those with matrilysin-2-negative cancer. Matrilysin-2 expression retained its significant predictive value for overall and disease-free survival in multivariate analysis. Moreover, patients with concomitant expression of matrilysin-2 and MMP-9 had the worst prognosis. Zymography revealed that matrilysin-2 expression was significantly correlated with expression of active MMP-9 in ESCC tissues. Matrilysin-2-transfected TE-1 ESCC cells showed active MMP-9 activity and were more invasive in vitro compared with mock-transfected TE-1 cells. The results of this study suggest that matrilysin-2, the expression of which is closely correlated with nuclear beta-catenin expression and active MMP-9 activity, plays a key role in the progression of ESCC.

Hedgehog-interacting protein (HIP) was identified as a putative antagonist of the hedgehog (Hh) pathway and as a target of Hh signaling. Our aim was to clarify the expression profiles and epigenetic alterations of the HIP gene in gastrointestinal cancer. The expression and promoter epigenetic status of HIP in cancer cell lines and freshly resected gastrointestinal cancer tissues were examined using RT-PCR, tissue microarray analysis, methylation-specific PCR, and chromatin immunoprecipitation assay. Cells were treated with the demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. WST-8 assays and In Vitro invasion assays after treatment with HIP-specific siRNA were performed. HIP expression levels were reduced in most of the gastrointestinal cancer cell lines and in a certain subset of cancer tissues, and these were correlated with promoter hypermethylation. A heterochromatic structure characterized by neither acetylated H3 nor acetylated H4, and histone H3 lysine 9 hypermethylation and histone H3 lysine 4 hypomethylation was observed in cancer cells in which the HIP gene was aberrantly silenced. On the other hand, overexpression of the HIP gene was also found in some cancer tissues and there were significant correlations between protein expression levels of HIP and those of Sonic hedgehog (Shh), Indian hedgehog, Patched, and glioma-associated oncogene homolog-1. An association was found between lymph node metastasis and HIP silencing in colorectal cancer tissues with strong Shh expression and between advanced TNM stage and HIP silencing in diffuse-type gastric cancer tissues with strong Shh expression. Down-regulation of HIP expression by siRNA resulted in a significant increase in colon cancer cell growth and invasion In Vitro. Silencing of the HIP gene due to hypermethylation and chromatin remodeling appears to be frequently involved in gastrointestinal tumorigenesis.

To clarify alterations of Dickkopfs (Dkks) and Kremen2 (Krm2) in gastrointestinal cancer.We investigated the expression profiles and epigenetic alterations of Dkks and Krm2 genes in gastrointestinal cancer using RT-PCR, tissue microarray analysis, and methylation specific PCR (MSP). Cancer cells were treated with the demethylating agent and/or histone deacetylase inhibitor. WST-8 assays and in vitro invasion assays after treatment with specific siRNA for those genes were performed.Dkks and Krm2 expression levels were reduced in a certain subset of the gastrointestinal cancer cell lines and cancer tissues. This was correlated with promoter hypermethylation. There were significant correlations between Dkks over-expression levels and beta-catenin over-expression in colorectal cancer. In colorectal cancers with beta-catenin over-expression, Dkk-1 expression levels were significantly lower in those with lymph node metastases than in those without. Down-regulation of Dkks expression by siRNA resulted in a significant increase in cancer cell growth and invasiveness in vitro.Down-regulation of the Dkks associated to promoter hypermethylation appears to be frequently involved in gastrointestinal tumorigenesis.