Katsuhiro Anami

Serum olfactomedin 4 (GW112, hGC‐1) in combination with Reg IV is a highly sensitive biomarker for gastric cancer patients

Gastric cancer (GC) is 1 of the most common human cancers. Early detection remains the most promising approach to improving long‐term survival of patients with GC. We previously performed Serial Analysis of Gene Expression (SAGE) on 4 primary GCs and identified several GC‐specific genes including Reg IV. Of these genes, olfactomedin 4 (OLFM4, also known as GW112 or hGC‐1) is a candidate gene for cancer‐specific expression. In this study, we examined the expression of olfactomedin 4 in human GC by immunohistochemistry. We also assessed serum olfactomedin 4 levels in GC patients by enzyme‐linked immunosorbent assay. 94 (56%) of 167 GC cases were positive for olfactomedin 4 by immunostaining. Olfactomedin 4 staining was observed more frequently in stage I/II cases than in stage III/IV cases. The serum olfactomedin 4 concentration in presurgical GC patients (n = 123, mean ± SE, 36.3 ± 3.5 ng/mL) was significantly higher than that in healthy individuals (n = 76, 16.6 ± 1.6 ng/mL). In patients with stage I GC, the sensitivity of serum olfactomedin 4 (25%) and Reg IV (35%) was superior to that of CA19‐9 (5%) or CEA (3%). Furthermore, in patients with stage I GC, the combination of olfactomedin 4 and Reg IV elevated the diagnostic sensitivity to 52%. These results suggest that serum olfactomedin 4 is a useful marker for GC and its measurement alone or in combination with Reg IV has utility in the early detection of GC.

Molecular pathology of gastric cancer: research and practice.

Recent advances in the understanding of molecular stomach carcinogenesis are reviewed. As to molecular events in individual mucin phenotypes of gastric cancer, the CDX2-Reg IV-SOX9 pathway is associated with the intestinal mucin phenotype, while OLFM4 and CLDN18 are novel markers for the gastric phenotype. microRNAs play an important role in epigenetic deregulation in gastric cancer. Many microRNAs are up-regulated and down-regulated, and some of these are associated with histological differentiation and cancer progression. Reduced miR-200 may participate in the genesis of diffuse type gastric cancer by reducing E-cadherin expression. Genetic polymorphism is a crucial endogenous cause and a fundamental factor of cancer risk. PSCA polymorphism alters the susceptibility to diffuse type gastric cancer through modulation of cell proliferation activity. Cancer stem cells possess the capacity for self-renewal and cause the heterogeneous lineages of cancer cells. Cancer stem cells also show resistance to anti-tumor chemotherapy. Only a minor population of gastric cancer cells reveals the properties of cancer stem cells, and CD44 is one of the markers for gastric cancer stem cells. The origin of gastric cancer stem cells remains to be elucidated.

High miR-21 expression from FFPE tissues is associated with poor survival and response to adjuvant chemotherapy in colon cancer.

Colon cancer (CC) is a leading cause of cancer mortality. Novel biomarkers are needed to identify CC patients at high risk of recurrence and those who may benefit from therapeutic intervention. The aim of this study is to investigate if miR‐21 expression from RNA isolated from formalin‐fixed paraffin‐embedded (FFPE) tissue sections is associated with prognosis and therapeutic outcome for patients with CC. The expression of miR‐21 was measured by quantitative reverse transcriptase‐polymerase chain reaction in a Japanese cohort (stage I‐IV, n = 156) and a German cohort (stage II, n = 145). High miR‐21 expression in tumors was associated with poor survival in both the stage II/III Japanese (p = 0.0008) and stage II German (p = 0.047) cohorts. These associations were independent of other clinical covariates in multivariable models. Receipt of adjuvant chemotherapy was not beneficial in patients with high miR‐21 in either cohort. In the Japanese cohort, high miR‐21 expression was significantly associated with poor therapeutic outcome (p = 0.0001) and adjuvant therapy was associated with improved survival in patients with low miR‐21 (p = 0.001). These results suggest that miR‐21 is a promising biomarker to identify patients with poor prognosis and can be accurately measured in FFPE tissues. The expression of miR‐21 may also identify patients who will benefit from adjuvant chemotherapy.

Noninvasive Urinary Metabolomic Profiling Identifies Diagnostic and Prognostic Markers in Lung Cancer

Lung cancer remains the most common cause of cancer deaths worldwide, yet there is currently a lack of diagnostic noninvasive biomarkers that could guide treatment decisions. Small molecules (<1,500 Da) were measured in urine collected from 469 patients with lung cancer and 536 population controls using unbiased liquid chromatography/mass spectrometry. Clinical putative diagnostic and prognostic biomarkers were validated by quantitation and normalized to creatinine levels at two different time points and further confirmed in an independent sample set, which comprises 80 cases and 78 population controls, with similar demographic and clinical characteristics when compared with the training set. Creatine riboside (IUPAC name: 2-{2-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-1-methylcarbamimidamido}acetic acid), a novel molecule identified in this study, and N-acetylneuraminic acid (NANA) were each significantly (P < 0.00001) elevated in non-small cell lung cancer and associated with worse prognosis [HR = 1.81 (P = 0.0002), and 1.54 (P = 0.025), respectively]. Creatine riboside was the strongest classifier of lung cancer status in all and stage I-II cases, important for early detection, and also associated with worse prognosis in stage I-II lung cancer (HR = 1.71, P = 0.048). All measurements were highly reproducible with intraclass correlation coefficients ranging from 0.82 to 0.99. Both metabolites were significantly (P < 0.03) enriched in tumor tissue compared with adjacent nontumor tissue (N = 48), thus revealing their direct association with tumor metabolism. Creatine riboside and NANA may be robust urinary clinical metabolomic markers that are elevated in tumor tissue and associated with early lung cancer diagnosis and worse prognosis.

Search for transmembrane protein in gastric cancer by the Escherichia coli ampicillin secretion trap: expression of DSC2 in gastric cancer with intestinal phenotype†

Gastric cancer (GC) is one of the most common malignancies worldwide. Genes expressed only in cancer tissue, and especially on the cell membrane, will be useful molecular markers for diagnosis and may also be good therapeutic targets. To identify genes that encode transmembrane proteins present in GC, we generated Escherichia coli ampicillin secretion trap (CAST) libraries from two GC cell lines and normal stomach. By sequencing 4320 colonies from CAST libraries, we identified 30 candidate genes that encode transmembrane proteins present in GC. Quantitative reverse transcription‐polymerase chain reaction analysis of these candidates revealed that ZDHHC14, BST2, DRAM2, and DSC2 were expressed much more highly in GC than in 14 kinds of normal tissues. Among these, DSC2 encodes desmocollin 2, which is one of three known desmocollins. Immunohistochemical analysis demonstrated that 22 (28%) of 80 GC cases were positive for desmocollin 2, and desmocollin 2 expression was observed frequently in GC with the intestinal mucin phenotype. Furthermore, desmocollin 2 expression was correlated with CDX2 expression. These results suggest that expression of desmocollin 2, induced by CDX2, may be a key regulator for GC with the intestinal mucin phenotype. Our results provide a list of genes that have high potential as a diagnostic and therapeutic target for GC. Copyright

Serial analysis of gene expression of esophageal squamous cell carcinoma: ADAMTS16 is upregulated in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. To identify potential diagnostic markers for ESCC and therapeutic targets for ESCC, we used Serial Analysis of Gene Expression (SAGE) on one ESCC sample. We obtained a total of 14 430 tags, including 5765 that were unique. By comparing SAGE tags from the ESCC sample with those from normal human squamous esophagus, we found several genes that were differentially expressed between ESCC and normal squamous esophagus. Among these, we focused on the ADAM metallopeptidase with thrombospondin type 1 motif, 16 (ADAMTS16) gene because quantitative RT‐PCR analysis showed a high level of ADAMTS16 expression in eight out of 20 ESCC samples (40%), but not in 15 kinds of normal tissues. Western blot analysis also showed upregulation of ADAMTS16 protein in ESCC tissues. Furthermore, ADAMTS16 protein was detected in culture media from the TE5 esophageal cancer cell line. Knockdown of ADAMTS16 in TE5 cells inhibited both cell growth and invasion ability. Our present SAGE data provide a list of genes potentially associated with ESCC. ADAMTS16 could be a novel diagnostic and therapeutic target for ESCC.

MicroRNA-155 is a predictive marker for survival in patients with clear cell renal cell carcinoma.

To investigate the clinical significance of micro‐ribonucleic acid‐155 in clear cell renal cell carcinoma, in particular focusing on the association of expression levels of micro‐ribonucleic acid‐155 with clinicopathological factors, cancer‐specific survival and therapeutic outcomes in clear cell renal cell carcinoma patients.

Immunohistochemical analysis of colorectal cancer with gastric phenotype: Claudin-18 is associated with poor prognosis

Claudin‐18 plays a key role in constructing tight junctions, and altered claudin‐18 expression has been documented in various human malignancies; however, little is known about the biological significance of claudin‐18 in colorectal cancer (CRC). The aim of this study is to investigate the significance of claudin‐18 expression in CRC and its association with clinicopathological factors. We performed clinicopathological analysis of claudin‐18 expression in a total of 569 CRCs by immunohistochemistry. Moreover, we investigated the association between claudin‐18 and various markers including gastric/intestinal phenotype (MUC5AC, MUC6, MUC2 and CD10), CDX2, claudin‐3, claudin‐4, p53 and Ki‐67.

Liver-intestine cadherin induction by epidermal growth factor receptor is associated with intestinal differentiation of gastric cancer.

Gastric cancer (GC) is one of the most common malignancies worldwide. The epidermal growth factor receptor (EGFR) molecule is very important in GC progression. To examine the correlation between EGFR and GC‐related genes, we analyzed gene expression profiles of HT‐29 cells treated with EGFR ligands and identified six genes upregulated by epidermal growth factor (EGF) and transforming growth factor (TGF)‐α treatment. Among these, we focused on cadherin 17 (CDH17) encoding liver–intestine cadherin (LI‐cadherin). Expression of LI‐cadherin was induced by both EGF and TGF‐α, as detected by quantitative RT‐PCR and Western blot analysis. A luciferase assay showed that LI‐cadherin promoter activity was enhanced by EGF or TGF‐α in both HT‐29 cells and MKN‐74 GC cells. Immunohistochemical analysis of 152 GC cases showed that out of 58 LI‐cadherin‐positive cases, 24 (41%) cases were also positive for EGFR, whereas out of 94 LI‐cadherin‐negative cases, only 9 (10%) cases were positive for EGFR (P < 0.0001). Double‐immunofluorescence staining revealed that EGFR and LI‐cadherin were coexpressed. Significant correlation was found between LI‐cadherin expression and advanced T grade and N grade. Both EGFR and LI‐cadherin expression were more frequently found in GC cases with an intestinal mucin phenotype than in cases with a gastric mucin phenotype. These results indicate that, in addition to the known intestinal transcription factor caudal type homeobox 2, EGFR activation induces LI‐cadherin expression and participates in intestinal differentiation of GC.

Increased MicroRNA-34b and -34c Predominantly Expressed in Stromal Tissues Is Associated with Poor Prognosis in Human Colon Cancer

The microRNA-34 family (miR-34a, -34b and -34c) have been reported to be tumor suppressor microRNAs (miRNAs) that are regulated by the TP53 and DNA hypermethylation. However, the expression, regulation, and prognostic value of the miR-34 family have not been systematically studied in colon cancer. To elucidate the roles of miR-34 family in colon carcinogenesis, miR-34a/b/c were measured in tumors and adjacent noncancerous tissues from 159 American and 113 Chinese colon cancer patients using quantitative RT-PCR, and we examined associations between miR-34a/b/c expression with TNM staging, cancer-specific mortality, TP53 mutation status and Affymetrix microarray data. All miR-34 family members were significantly increased in colon tumors, counter to the proposed tumor suppressor role for these miRNAs. Increased miR-34b/c were observed in more advanced tumors in two independent cohorts and increased expression of miR-34b/c was associated with poor cancer-specific mortality. While the expression of miR-34 family was not associated with TP53 mutation status, TP53 transcriptional activity was associated with miR-34a/b/c expression that is consistent with the proposed regulation of miR-34a/b/c by TP53. To examine where the miR-34 family is expressed, the expression of miR-34 family was compared between epitheliums and stromal tissues using laser microdissection technique. The expression of miR-34b/c was increased significantly in stromal tissues, especially in cancer stroma, compared with epithelial tissue. In conclusion, increased miR-34b/c predominantly expressed in stromal tissues is associated with poor prognosis in colon cancer. MiR-34 may contribute to cancer-stromal interaction associated with colon cancer progression.