Shigeki Tomita

Expression of SDF-1α and nuclear CXCR4 predicts lymph node metastasis in colorectal cancer

Although stromal cell-derived factor (SDF)-1α and its receptor CXCR4 are experimentally suggested to be involved in tumorigenicity, the clinicopathological significance of their expression in human disease is not fully understood. We examined SDF-1α and CXCR4 expression in colorectal cancers (CRCs) and their related lymph nodes (LNs), and investigated its relationship to clinicopathological features. Specimens of 60 primary CRCs and 27 related LNs were examined immunohistochemically for not only positivity but also immunostaining patterns for SDF-1α and CXCR4. The relationships between clinicopathological features and SDF-1α or CXCR4 expression were then analysed. Stromal cell-derived factor-1α and CXCR4 expression were significantly associated with LN metastasis, tumour stage, and survival of CRC patients. Twenty-nine of 47 CXCR4-positive CRCs (61.7%) showed clear CXCR4 immunoreactivity in the nucleus and a weak signal in the cytoplasm (nuclear type), whereas others showed no nuclear immunoreactivity but a diffuse signal in the cytoplasm and at the plasma membrane (cytomembrane type). Colorectal cancer patients with nuclear CXCR4 expression showed significantly more frequent LN metastasis than did those with cytomembrane expression. Colorectal cancer patients with nuclear CXCR4 expression in the primary lesion frequently had cytomembrane CXCR4-positive tumours in their LNs. In conclusion, expression of SDF-1α and nuclear CXCR4 predicts LN metastasis in CRCs.

Nuclear expression of phosphorylated EGFR is associated with poor prognosis of patients with esophageal squamous cell carcinoma.

Objectives: Although it has been reported that epidermal growth factor receptor (EGFR) is able to translocate from the plasma membrane to the nucleus, the pathophysiological role of this translocation in tumorigenicity is still unclear. In the present study, to elucidate the pathophysiological significance of EGFR translocation, we investigated the expression not only of conventional EGFR but also its phosphorylated form (pEGFR), focusing on its cellular localization in esophageal cancer tissues. Methods: Fifty-two specimens of esophageal squamous cell carcinoma (SCC) obtained by surgery were examined immunohistochemically for their EGFR and pEGFR immunostaining patterns. The relationships between clinicopathological parameters and EGFR or pEGFR immunostaining patterns were then analyzed. Results: In 37 (71.2%) of the 52 esophageal SCCs, EGFR immunoreactivity was clearly localized at the plasma membrane of the cancer cells, whereas pEGFR immunoreactivity was clearly localized in the nucleus in 19 (36.5%) cases. Nuclear expression of pEGFR significantly correlated with TNM stage and lymph node metastasis, and moreover was associated with a poor outcome of esophageal SCC. Conclusions: Nuclear translocalization of pEGFR is associated with an increase in the malignant potential of esophageal SCC and may affect prognosis in patients with esophageal SCC.

Involvement of the IL-22/REG Iα axis in ulcerative colitis

The Regenerating gene (REG) Iα protein, a trophic and/or anti-apoptotic factor, is important in the pathophysiology of gastrointestinal inflammation. Interleukin (IL)-22 is a recently identified cytokine that is suggested to have pivotal roles in inflammatory bowel diseases. We therefore investigated the involvement of the IL-22/REG Iα axis and examined the mechanism of regulation of REG Iα expression by IL-22 stimulation in ulcerative colitis (UC) mucosa. Expression of IL-22, IL-22 receptor 1 (IL-22R1), and REG Iα in UC mucosa was analyzed by real-time RT-PCR and immunohistochemistry. The effects of IL-22 on REG Iα protein expression were examined using a small-interfering RNA for STAT3, an MAPK inhibitor or a PI3K inhibitor. The element responsible for IL-22-induced REG Iα promoter activation was determined by a promoter deletion and electrophoretic mobility shift assay. The expression of IL-22 was enhanced in infiltrating inflammatory cells, and that of IL-22R1 and REG Iα was concurrently enhanced in the inflamed epithelium in UC mucosa. The levels of REG Iα and IL-22 mRNA expression were strongly correlated, and the distributions of REG Iα- and IL-22R1-positive epithelial cells were very similar. IL-22 simulation enhanced the expression of REG Iα protein through STAT3 tyrosine phosphorylation in colon cancer cells. The IL-22-responsive element was located between −142 and −134 in the REG Iα promoter region. REG Iα protein may have a pathophysiological role as a biological mediator for immune cell-derived IL-22 in the UC mucosa.

Methylation of the oestrogen receptor gene in non-neoplastic epithelium as a marker of colorectal neoplasia risk in longstanding and extensive ulcerative colitis

Background: Surveillance colonoscopy is widely recommended in patients with longstanding and extensive ulcerative colitis (UC) in order to detect colorectal neoplasia at an early stage. However, it still remains questionable whether surveillance colonoscopy effectively enables early detection of UC associated neoplasia. There is a great need for sensitive markers to identify individuals at increased risk of neoplasia. The oestrogen receptor (OR) gene shows age related methylation in the colorectal epithelium and is methylated frequently in sporadic colorectal neoplasia, suggesting that OR methylation may predispose to colorectal neoplasia. Aim: To clarify whether analysis of methylation of the OR gene in non-neoplastic epithelium can contribute to prediction of increased neoplasia risk in UC patients. Materials and methods: A total of 165 non-neoplastic colorectal epithelia from 30 patients with longstanding and extensive UC, including 13 UC patients with neoplasia and 17 patients without, were evaluated. Methylation specific polymerase chain reaction was performed to determine the methylation status of the OR gene. Results: Methylation of the OR gene was detected in 54 of 70 (77.1%) non-neoplastic colorectal epithelia in UC with neoplasia but in only 23 of 95 (24.2%) without neoplasia. Methylation of the OR gene was significantly more frequent in non-neoplastic epithelium from UC with neoplasia than in chronic colitic epithelium from UC without neoplasia. Furthermore, in UC with neoplasia, the OR gene was extensively methylated in non-neoplastic epithelia throughout the colorectum compared with those in UC without neoplasia. Conclusion: These results suggest that analysis of OR gene methylation may have potential as a useful marker for identifying individuals at increased risk of neoplasia among those with longstanding and extensive UC.

Clinical validation of the gastrointestinal NET grading system: Ki67 index criteria of the WHO 2010 classification is appropriate to predict metastasis or recurrence.

BackgroundIn the WHO 2010 classification, the neuroendocrine tumors (NETs) are subdivided by their mitotic index or Ki67 index into either G1 or G2 NETs. Tumors with a Ki67 index of <2% are classified as G1 and those with 3—20% are classified as G2. However, the assessment of tumors with Ki67 index of greater than 2% and less than or equal to 3% is still unclear. To resolve the problem, we validated the Ki67 index criteria of gastrointestinal NETs of the WHO 2010 classification.MethodsThe medical records of 45 patients who were pathologically diagnosed as having NET G1/G2 of the gastrointestinal tract were analyzed retrospectively. According to the WHO 2010 classification, Ki67 index were calculated. Computer-assisted cytometrical analysis of Ki67 immunoreactivity was performed using the WinRooF image processing software. Receiver operating characteristic (ROC) curves were generated to determine the best discriminating Ki67 index. To clarify the assessment of tumors with Ki67 index between 2—3%, the calculated cutoff of Ki67 index was evaluated using Fisher’s exact test.ResultsROC curve analysis confirmed that 2.8% was the best Ki67 index cutoff value for predicting metastasis or recurrence. The sensitivity of the new Ki67 index cutoff was 42.9%, and the specificity was 86.8%.ConclusionsDivision of NETs into G1/G2 based on Ki67 index of 3% was appropriate to predict metastases or recurrences. The WHO grading system may be the most useful classification to predict metastases or recurrences.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1553036118943799

Prediction of Colorectal Neoplasia by Quantitative Methylation Analysis of Estrogen Receptor Gene in Nonneoplastic Epithelium from Patients with Ulcerative Colitis

Purpose: The incidence of colorectal neoplasia has increased among patients with longstanding and extensive ulcerative colitis (UC). Therefore, surveillance colonoscopy has been widely recommended. However, there is controversy about the impact of cancer surveillance, and ways to improve its effectiveness are being sought. The estrogen receptor (ER) gene shows age-related methylation in the colorectal epithelium and is frequently methylated in colorectal neoplasia, suggesting that ER methylation occurs early in the process of colorectal tumorigenesis. Experimental Design: To clarify whether methylation analysis of the ER gene in nonneoplastic epithelium can help predict an increased risk for UC-associated neoplasia, a total of 105 nonneoplastic colorectal epithelia from 18 patients with longstanding and extensive UC, including 8 patients with neoplasia and 10 patients without neoplasia, were analyzed. In all patients, multiple samples were taken from six regions of the colorectum. The combined bisulfite restriction analysis method was used to determine the methylation status of the ER gene. Results: The mean methylation level of the ER gene was 25.4% in the nonneoplastic epithelia from UC patients with neoplasia, whereas it was only 4.0% in those without neoplasia (P < 0.001). The methylation level of the ER gene in UC patients with neoplasia was significantly higher than in UC patients without neoplasia throughout the colorectum except for the cecum. In UC patients with neoplasia, the mean ER methylation level in the distal colon (36.1%) was significantly higher than in the proximal colon (14.6%; P < 0.001). Conclusions: These results suggest that the analysis of ER gene methylation in nonneoplastic colorectal epithelium could have the potential to be a useful adjunct for identifying individuals with longstanding and extensive UC who are at increased risk of neoplasia and contribute to more effective cancer surveillance.

DMBT1 is a novel gene induced by IL‐22 in ulcerative colitis

Background: Interleukin (IL)‐22 is a recently identified cytokine that is suggested to play pivotal roles in various inflammatory diseases. Although the IL‐22 receptor 1 (IL‐22R1) is restrictively expressed in epithelial cells in the colon, the role of IL‐22 in colonic diseases still remains unclear. In this study microarray analyses revealed that deleted in malignant brain tumors 1 (DMBT1) is a novel upregulated gene in IL‐22‐stimulated colon cancer cells. Therefore, we investigated the involvement of DMBT1 and IL‐22 in ulcerative colitis (UC) tissues and examined the mechanism regulating the expression of DMBT1 in response to IL‐22 stimulation. Methods: Changes of gene expression in IL‐22‐stimulated SW403 cells were investigated by microarray analyses. The effects of IL‐22 on DMBT1 expression were examined in SW403 cells using a small interfering RNA (si)RNA for STAT3 or inhibitors for MEK, PI3K, and nuclear factor kappa B (NF‐&kgr;B). The element responsible for IL‐22‐induced DMBT1 promoter activation was determined by a promoter deletion and electrophoretic mobility shift assay (EMSA). Expression of IL‐22, IL‐22R1, and DMBT1 in UC tissues was analyzed by real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) and immunohistochemistry. Results: IL‐22 treatment enhanced the expression of DMBT1 through STAT3 tyrosine phosphorylation and NF‐&kgr;B activation in colon cancer cells. The IL‐22‐responsive element was located between −187 and −179 in the DMBT1 promoter region. In the UC mucosa the levels of DMBT1 and IL‐22 mRNA expression were significantly enhanced and positively correlated, the numbers of IL‐22‐positive lymphocytes were increased, and the expression of IL‐22R1 and DMBT1 was enhanced in the inflamed epithelium. Conclusions: The IL‐22/DMBT1 axis may play a pivotal role in the pathophysiology of UC. (Inflamm Bowel Dis 2010;)

Expression profile of REG family proteins REG Iα and REG IV in advanced gastric cancer: Comparison with mucin phenotype and prognostic markers

Regenerating gene family members 1 (REG Iα) and 4 (REG IV) are overexpressed in a subset of gastric cancers. However, comparative characterization of the expression of these family proteins has remained unclear. Therefore, we aimed to elucidate not only the association between REG protein expression and mucin phenotype but also their significance as a prognostic marker for patients with gastric cancer. The expression of REG Iα, REG IV, CDX2, MUC2, and MUC5AC in gastric cancer tissues was examined by immunohistochemistry. The relationship between REG protein expression and clinicopathological parameters or mucin phenotype was then analyzed. REG Iα and REG IV expression was positive in 33 (52%) and 31 (49%) of 63 gastric cancers examined, respectively. REG Iα expression was significantly related to venous invasion and tumor stage, whereas REG IV expression showed no relationship to clinicopathological features. With regard to mucin phenotype, REG IV expression was significantly correlated with MUC2 and CDX2 expression, suggesting an association with the intestinal mucin phenotype of gastric cancer. On the other hand, REG Iα expression had no correlation with MUC2, CDX2, or MUC5AC in gastric cancer tissues. Expression of REG Iα but not REG IV was an independent predictor of poor outcome in patients with gastric cancer. In addition, patients with gastric cancer negative for both REG Iα and REG IV expression had a significantly better outcome than patients positive for either REG Iα or REG IV. Profiling of REG protein expression is useful to for prognostication of patients with gastric cancer.

Analysis of K-ras mutations and expression of cyclooxygenase-2 and gastrin protein in laterally spreading tumors.

Background and Aim: Recent advances in colonoscopic techniques have led to the increased detection of, and interest in, superficial type colorectal tumors, and a new category, the ‘laterally spreading tumor (LST)’, has been proposed. However, the characteristics of the genetic alterations in these LSTs have not yet been fully determined. We therefore classified LSTs as LST‐granular (LST‐G) or LST‐non‐granular (LST‐NG), according to their macroscopic appearance, and examined the genetic alterations in these two tumor groups compared with those in protruded type tumors.

Evaluation of Tumor Cell Dissociation as a Predictive Marker of Lymph Node Metastasis in Submucosal Invasive Colorectal Carcinoma

PURPOSETumor cell dissociation—the histologic finding of small solid carcinoma cell clusters and groups of dissociated dedifferentiated carcinoma cells at the invasive front–is related to tumor metastasis and patient prognosis. However, few previous reports have examined tumor cell dissociation in submucosal invasive colorectal carcinoma. We investigated the relation between tumor cell dissociation and lymph node metastasis in submucosal invasive colorectal carcinoma. We also examined immunohistochemical expression of E-cadherin and beta-catenin in submucosal invasive colorectal carcinoma.METHODSSubmucosal invasive colorectal carcinoma tissue samples from 20 patients with lymph node metastasis and 100 patients without lymph node metastasis were evaluated. Sections stained with hematoxylin and eosin were evaluated for tumor cell dissociation. Immunohistochemistry was used to determine the expression and cellular distribution of E-cadherin and beta-catenin.RESULTSTumor cell dissociation was more frequently identified in submucosal invasive colorectal carcinoma cases with lymph node metastasis than in those without lymph node metastasis (P = 0.0001). Decreased membranous expression of E-cadherin occurred more frequently in submucosal invasive colorectal carcinoma cases with lymph node metastasis than in those without lymph node metastasis (P = 0.025). Nuclear expression of beta-catenin tended to be present in submucosal invasive colorectal carcinoma cases with lymph node metastasis (P = 0.063). Decreased membranous expression of E-cadherin occurred more frequently in submucosal invasive colorectal carcinoma cases with tumor cell dissociation than in those without tumor cell dissociation (P = 0.0023).CONCLUSIONSOur results suggest that there is a relation between tumor cell dissociation and lymph node metastasis in submucosal invasive colorectal carcinoma. Tumor cell dissociation formation might be related to abnormal expression patterns of E-cadherin and beta-catenin in submucosal invasive colorectal carcinoma. Tumor cell dissociation and decreased membranous expression of E-cadherin would be important predictive markers for lymph node metastasis in submucosal invasive colorectal carcinoma.