Mikito Mori

Serum microRNA expression profile: miR-1246 as a novel diagnostic and prognostic biomarker for oesophageal squamous cell carcinoma

Background:Recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in blood and can serve as useful biomarkers for cancer.Methods:We performed an miRNA array using serum samples obtained from oesophageal squamous cell carcinoma (ESCC) patients or healthy controls. MiR-1246 was the most markedly elevated in ESCC patients. Therefore, miR-1246 was selected as a candidate for further analysis. The serum miR-1246 level in 46 healthy controls and 101 ESCC patients was evaluated and compared among various clinicopathological characteristics. MiR-1246 expressions in tissue, exosomal, and cellular samples were also examined.Results:Serum miR-1246 alone yielded an receiver-operating characteristic curve area of 0.754, with 71.3% sensitivity and 73.9% specificity for distinguishing ESCC patients from healthy controls. Serum miR-1246 was significantly correlated with the TNM stage and showed to be the strongest independent risk factor for poor survival (HR, 4.032; P=0.017). Unlike the tendency shown in previous reports, miR-1246 was not upregulated in ESCC tissue samples. Furthermore, exosomal miR-1246 did not reflect the abundance in the cell of origin.Conclusion:These data support our contention that serum miR-1246 has strong potential as a novel diagnostic and prognostic biomarker in ESCC, and its releasing mechanism is selective and independent of tissue miRNA abundance.

Sentinel lymph node mapping for gastric cancer using a dual procedure with dye- and gamma probe-guided techniques

BACKGROUND Increasing evidence supports the sentinel lymph node (SN) concept for melanoma and breast cancers. SN biopsy may replace routine lymph node dissection in the treatment of these cancers. But there are little data evaluating this concept in patients with gastric cancer. The objective of this study was to test the feasibility of SN mapping in gastric cancers by using the dual-mapping procedure with dye and radioactive colloid. STUDY DESIGN Thirty-one consecutive patients preoperatively diagnosed as T1-2 and N0 underwent SN biopsy using the dual-mapping procedure. Distributions of SNs identified by the dye-guided technique (blue nodes; BNs) were compared with those identified by the gamma probe guided technique (hot nodes; HNs). RESULTS Among the 31 patients, 7 were found to have lymph node metastases. All positive nodes were detected by SN biopsy using the dual method. So, an accuracy rate of 100% was achieved in predicting the status of regional lymph nodes. Both BNs and HNs were identified in 28 of 31 patients (90%), but significant discrepancy of distribution was noted between BNs and HNs. Among the 28 patients with identified BNs, there was one metastasis in a non-BN. So the accuracy rate was 96% for the dye-guided technique. In contrast, among the 28 patients with identified HNs, 2 patients had metastasis in non-HNs, making the accuracy rate 93% for the gamma probe-guided technique. CONCLUSIONS SN mapping is feasible in gastric cancer, but the dye-guided and gamma probe-guided techniques are complementary. So we recommend the dual-mapping procedure.

Identification of novel molecular targets regulated by tumor suppressive miR-375 induced by histone acetylation in esophageal squamous cell carcinoma

The aim of this study was to determine whether histone acetylation regulates tumor suppressive microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC) and to identify genes which are regulated by these miRNAs. We identified a miRNA that was highly upregulated in an ESCC cell line by cyclic hydroxamic acid-containing peptide 31 (CHAP31), one of the histone deacetylase inhibitors (HDACIs), using a miRNA array analysis. miR-375 was strongly upregulated by CHAP31 treatment in an ESCC cell line. The expression levels of the most upregulated miRNA, miR-375 were analyzed by quantitative real-time PCR in human ESCC specimens. The tumor suppressive function of miR-375 was revealed by restoration of miR-375 in ESCC cell lines. We performed a microarray analysis to identify target genes of miR-375. The mRNA and protein expression levels of these genes were verified in ESCC clinical specimens. LDHB and AEG-1/MTDH were detected as miR‑375-targeted genes. The restoration of miR-375 suppressed the expression of LDHB and AEG-1/MTDH. The ESCC clinical specimens exhibited a high level of LDHB expression at both the mRNA and protein levels. A loss-of-function assay using a siRNA analysis was performed to examine the oncogenic function of the gene. Knockdown of LDHB by RNAi showed a tumor suppressive function in the ESCC cells. The correlation between gene expression and clinicopathological features was investigated by immunohistochemistry for 94 cases of ESCC. The positive staining of LDHB correlated significantly with lymph node metastasis and tumor stage. It also had a tendency to be associated with a poor prognosis. Our results indicate that HDACIs upregulate miRNAs, at least some of which act as tumor suppressors. LDHB, which is regulated by the tumor suppressive miR-375, may therefore act as an oncogene in ESCC.

Histone Deacetylase Inhibitor FK228 Activates Tumor Suppressor Prdx1 with Apoptosis Induction in Esophageal Cancer Cells

Purpose: The histone deacetylase inhibitor FK228 shows strong activity as a potent antitumor drug but its precise mechanism is still obscure. The purpose of this study is to reveal the effect of FK228 on gene expression in the cell and to determine the mechanism of the antitumor activity of FK228 for further clinical applications. Experimental Design and Results: Microarray analysis was applied to verify the gene expression profiles of 4,608 genes after FK228 treatment using human esophageal squamous cell cancer cell lines T.Tn and TE2. Among them, peroxiredoxin 1 (Prdx1), a member of the peroxiredoxin family of antioxidant enzymes having cell growth suppression activity, as well as p21WAF1, were significantly activated by FK288. In addition, FK228 strongly inhibited the cell growth of T.Tn and TE2 by the induction of apoptosis. Further, chromatin immunoprecipitation analysis revealed that FK228 induced the accumulation of acetylated histones H3 and H4 in Prdx1 promoter, including the Sp1-binding site. In mouse xenograft models of T.Tn and TE2 cells, FK228 injection resulted in significant tumor regression as well as activated Prdx1 expression in tumor tissues. Prdx1 suppression by RNA interference hindered the antitumor effect of FK228. Conclusion: Our results indicate that the antitumor effect of FK228 in esophageal cancer cells is shown at least in part through Prdx1 activation by modulating acetylation of histones in the promoter, resulting in tumor growth inhibition with apoptosis induction.

Clinical and Pathologic Evaluation of the Effectiveness of Neoadjuvant Chemoradiation Therapy in Advanced Esophageal Cancer Patients

BackgroundChemoradiation therapy (CRT) has the strongest antitumor effect against local tumors of esophageal cancer; however, no standard strategy has yet been established to achieve a clinical complete response (CR) after CRT. The aim of this study was to clarify when a decision can be made to perform further treatment for a clinical CR.MethodsWe evaluated 78 patients that underwent an esophagectomy after neoadjuvant CRT in our department between 1998 and 2007. The study investigated the clinical and pathologic results of neoadjuvant CRT.ResultsOf the 78 cases, 19 (24.3%) were a pathologic CR (Grade 3). Pathologic CR could be estimated in only 3 of 8 clinical CR cases (37.5%). On the other hand, 12 (20.7%) of the 58 clinical partial response (PR) cases achieved pathologic CR. Likewise, 4 cases (36.4%) achieved pathologic CR among the clinical no change/progressive disease (NC/PD) patients.ConclusionsThe clinical evaluation for CRT does not reflect the pathologic effectiveness and, even if clinical CR was achieved, viable cancer cells were still present at the primary site in the majority of the population.

Surgical advantages of gastric SMTs by laparoscopy and endoscopy cooperative surgery.

BACKGROUND/AIMS The treatment of gastric submucosal tumors (SMTs) is strictly surgical and enucleation of the tumor or wedge resection of the stomach is efficient to achieve R0 resection. Laparoscopic and endoscopic cooperative surgery (LECS) can be safely performed with adequate cutting lines. This study describes the initial 16 cases treated by LECS and evaluates the advantages by LECS for gastric SMTs retrospectively. METHODOLOGY Sixteen patients with gastric SMT underwent LECS from June 2007 to December 2010, their surgical data, clinical characteristics and surgical specimens of SMTs were compared. The surgical specimens of 9 gastric SMTs treated by laparoscopic wedge resection (LWR) were compared as a control. RESULTS The median (range) length of operation time, blood loss, hospital stay after surgery were minutes 172 (115- 220), <5mL (<5-115) and 10 days (6-17), respectively. The median (range) ratio of the longest diameter of the tumor divided by the longest diameter of the surgical specimen in LECS and LWR were 0.86 (0.625-1.0) and 0.69 (0.44-1.0), respectively (p=0.0189, Wilcoxon rank sum test). CONCLUSIONS LECS minimizes the surgical specimen while still providing sufficient surgical margins to successfully cure gastric SMTs.

A COX-2 inhibitor enhances the antitumor effects of chemotherapy and radiotherapy for esophageal squamous cell carcinoma

Cyclooxygenase-2 (COX-2) is a key enzyme of prostaglandin (PG) synthesis that has been demonstrated to be overexpressed in several types of cancers. The function of COX-2 in tumor progression has been recently elucidated. In tumors in which COX-2 is overexpressed, the antitumor effects are suppressed. We examined the effects of celecoxib, a COX-2 inhibitor, in enhancing the antitumor effects of chemotherapy and radiotherapy for esophageal squamous cell carcinoma (ESCC) by reducing the COX-2 activity. We used the human esophageal squamous cell lines TE2 and T.Tn treated with celecoxib and 5-FU/radiation, after which cell viability assays were performed. Changes in the expressions of dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT) mRNA and PGE2 were also measured. In addition, apoptotic changes, and the invasion and migration activity in both the celecoxib and 5-FU treated cells were evaluated. The experiments showed that T.Tn and TE2 proliferation was strongly inhibited by the combination of 5-FU/radiation and the COX-2 inhibitor. Inhibiting the COX-2 activity induced a reduction in PGE2 levels in TE2/T.Tn cells. Following treatment with the COX-2 inhibitor and 5-FU, the OPRT expression was upregulated and the DPD expression was downregulated in the resistant cells. In addition, the combination treatment with the COX-2 inhibitor and 5-FU markedly inhibited both the cell invasion and migration activity. Therefore, COX-2 inhibitors can be useful enhancers of antitumor drugs and radiotherapy for ESCC.

Modulated electro-hyperthermia enhances dendritic cell therapy through an abscopal effect in mice

The aim of this study was to assess whether modulated electro-hyperthermia (mEHT) can induce an abscopal effect and thereby enhance the antitumor effects of immunotherapy. We used an intratumoral dendritic cell (DC) injection and mEHT to treat C3H/He mice inoculated with squamous cell carcinoma SCCVII cells in the left leg, and we assessed the whole body antitumor effects. Tumors were examined every two or three days in order to assess growth inhibition. The tumor-draining lymph nodes were removed to enable flow cytometric analysis of CD3+ and CD8+ cells, whereas immunohistochemistry was used to assess CD8, S100 and Foxp3 expression in the tumors. Additionally, GP96 expression in the tumors from the different treatment groups was measured. In the control group, the mean tumor volume was larger than that in other groups. These results indicated that the combination therapy of an intratumoral DC injection and mEHT evoked systemic antitumor activity. A larger number of CD3+ and CD8+ cells were detected by flow cytometric analysis in the DC plus mEHT treatment group. Tumor tissue immunostaining showed that CD8 and S100 were more strongly expressed in the DC plus mEHT treatment group, although Foxp3 expression was much higher in the control group. The GP96 gene expression level in the mEHT group was significantly different from the expression level in the control group. An abscopal effect may be induced by mEHT, and the effect of immunotherapy with DCs was strongly enhanced by the overexpression of GP96. GP96 is thought to be one of the molecules explaining the abscopal effect. Direct intratumoral administration of DCs and mEHT may be a feasible future treatment strategy.

Neoadjuvant chemoradiotherapy followed by esophagectomy versus definitive chemoradiotherapy in resectable stage II/III (T1-3N0, 1M0) esophageal squamous cell carcinoma

BackgroundThere are two intensive modalities for the treatment of resectable esophageal carcinoma: esophagectomy and definitive chemoradiotherapy (CRT). Esophagectomy with preoperative CRT was retrospectively compared with CRT alone in resectable stage II/III esophageal squamous cell carcinoma.MethodsSeventy-four patients with resectable stage II/III (T1-3N0, 1M0) esophageal squamous cell carcinoma were treated with preoperative CRT by 5-fluorouracil (5-FU) 700 mg/m2 on days 1 to 5, nedaplatin 80 mg/m2 on day 1, and concurrent radiation for a total of 30 Gy in 3 weeks. If patients decided to undergo surgery, esophagectomy from the thoracoabdominal approach was carried out 6 weeks after the completion of CRT (CRT + Surg group, n = 51). If patients decided not to undergo surgery, they were treated with one more course of CRT (CRT-alone group, n = 23).ResultsThere was no significant difference in overall survival between the two groups (P = 0.1006), whereas the disease-free survival in the CRT + Surg group was improved compared with the CRT-alone group (P = 0.0186). In the patients with clinical stage III carcinoma or with regional lymph node metastasis, the overall survival rate was significantly improved in the CRT + Surg group compared with the CRT-alone group. The rate of local failures in the CRT + Surg group was significantly lower compared with the CRT-alone group (P = 0.0011).ConclusionsPreoperative CRT followed by esophagectomy provides better local control, but does not prolong overall survival, compared with definitive CRT. However, in clinical stage III or N1, esophagectomy with preoperative CRT could contribute to the improvement of survival in esophageal squamous cell carcinoma.

Usefulness of microRNA‑375 as a prognostic and therapeutic tool in esophageal squamous cell carcinoma.

The aim of this study was to clarify the importance of microRNA‑375 (miR‑375) expression in patients with esophageal squamous cell carcinoma (ESCC) and to examine the in vivo antitumor effects of miR‑375 in a model of ESCC using a non‑viral delivery system. We estimated the miR‑375 and LDHB and AEG‑1/MTDH mRNA expression of the ESCC tumors from 85 patients. The correlation between the miR‑375 expression and clinicopathological features, including the prognosis, were evaluated. The presence of high miR‑375 expression was associated with lymphatic vessel invasion, while a low expression of miR‑375 significantly correlated with a poor prognosis for the 85 ESCC patients. We also found that there was a significant inverse correlation between the expression of miR‑375 and that of LDHB. Before the examination of miR‑375 in the in vivo assay, we confirmed that atelocollagen prolonged the accumulation of miRNA by using fluorescently‑labeled miRNA and an in vivo imaging system. We injected the miR‑375/atelocollagen complex or a control‑miRNA/atelocollagen complex into mice bearing TE2 and T.Tn xenografts via subcutaneous (s.c.) injections. The growth of both the TE2 and T.Tn tumors in the miR‑375 groups was significantly suppressed compared with that in the control‑miRNA groups. In addition, The LDHB mRNA expression of TE2 xenografts was significantly downregulated after miR‑375 treatment. In conclusion, it might be possible for the level of miR‑375 expression to be a utilized as a prognostic indicator for ESCC patients. The administration of miR‑375 using a non‑viral delivery might represent a powerful new treatment for ESCC.