The Risk of Metastatic Recurrence after Non-Curative Endoscopic Resection with Negative Deep Margins for Early Colorectal Cancer: Two-Center Retrospective Cohort Study.

INTRODUCTION: Non-curative endoscopic resection of T1 colorectal cancer (CRC) carries a substantial risk of recurrence. However, previous studies have reported a significant proportion of cases in which the deep margin of endoscopic resection was positive for cancer due to the technical difficulties of colorectal endoscopic submucosal dissection (ESD). With the advancement of endoscopic technology and techniques resulting in the reduction of positive resection margins, it is important to reassess the long-term prognosis and major risk factors for recurrence in cases of negative deep margins. METHODS: We conducted a retrospective cohort study of consecutive patients with T1 CRC who underwent endoscopic resection between January 2006 and December 2021 with negative deep margins. The histological findings of the resected specimens were analyzed to determine the risk factors associated with the primary outcomes of this study, including recurrence and cancer-related deaths. RESULTS: The median age of the 190 patients was 70 years, of which 63% were male, and endoscopic treatment was performed in 64% by endoscopic mucosal resection and 36% by ESD. Eighty-two patients were in the curative resection (CR) group and 108 were in the non-curative resection (NCR) group, wherein the latter comprised 79 patients who underwent additional surgery (AS) and 29 patients who did not receive AS. Five-year recurrence-free survival rates were 98.4% (95% CI: 89.3-99.8) for CR, 98.3% (95% CI: 88.8-99.8) for NCR with AS, and 73.7% (95% CI: 46.5-88.5) for NCR without AS. Lymphatic invasion and budding grade 2/3 were the major risk factors for recurrence, with hazard ratios of 40.7 (p < 0.001) and 23.1 (p = 0.007), respectively. Of the patients in the NCR group without AS, the 5-year recurrence-free rate was 85.6% (95% CI: 52.5-96.3) if there were no major risk factors (i.e., no lymphatic invasion or budding grade 2/3) (n = 21), whereas the prognosis was poor in the presence of one or more of the major risk factors, with a median recurrence-free survival and disease-specific survival of 2.5 and 3.1 years, respectively (n = 8). DISCUSSION: In endoscopically resected T1 CRC with negative deep margins, lymphatic invasion or budding grade 2/3 may indicate a higher risk of recurrence when followed up without AS.

Results of the interim analysis of a prospective, multicenter, observational study of small subepithelial lesions in the stomach.

OBJECTIVES: Long-term outcomes of gastric subepithelial lesions have not been elucidated. To reveal the natural history, we initiated a prospective, 10-year follow-up of patients with small (≤20 mm) gastric subepithelial lesions in September 2014. Here, we report the results of an interim analysis of a prospective observational study. METHODS: In total, 567 patients with 610 lesions were prospectively registered between September 2014 and August 2016. The location, size, morphology, and number of subepithelial lesions were recorded on a web-based case report form. This study has been conducted as an Academic Committee Working Group of the Japan Gastroenterological Endoscopy Society. RESULTS: The endoscopic follow-up period was 4.60 ± 1.73 years (mean ± standard deviation), and survival data were investigated for 5.28 ± 1.68 years. This interim analysis revealed that the estimated cumulative incidence of a size increase ≥5 mm, after accounting for patients' death and resection of the tumor as competing risk events, was 4.5% at 5 years. In addition, the estimated cumulative incidence of lesion size increase ≥5 mm or resection of lesions was 7.9% at 5 years, and that of size increase ≥10 mm or resection of lesions was 4.5% at 5 years. CONCLUSION: These results indicate that approximately one in 13 patients with small (≤20 mm) gastric subepithelial lesions may require resection or further investigation for increased tumor size (≥5 mm) within 5 years.

Long-term outcomes of patients with primary intestinal follicular lymphoma managed with watch-and-wait strategy.

Patients with primary intestinal follicular lymphoma are often followed-up without a specific treatment, and this approach is called the "watch-and-wait approach." However, the long-term outcomes of this patient group have not been sufficiently investigated. We enrolled patients with primary intestinal follicular lymphoma who were diagnosed before 2016 and managed with the watch-and-wait approach in 20 institutions. We retrospectively investigated the overall, disease-specific, and event-free survival rates as well as the rate of spontaneous regression. Among the 248 patients with follicular lymphoma with gastrointestinal involvement, 124 had localized disease (stage I or II1). We analyzed the data of 73 patients who were managed using the watch-and-wait approach. During the mean follow-up period of 8.3 years, the follicular lymphoma had spontaneously resolved in 16.4% of the patients. The 5-year and 10-year overall survival rates were 92.9% and 87.1%, respectively. With disease progression (n = 7), initiation of therapy (n = 7), and histologic transformation to aggressive lymphoma (n = 0) defined as events, the 5-year and 10-year event-free survival rates were 91.1% and 86.9%, respectively. No patient died of progressive lymphoma. Thus, both 5-year and 10-year disease-specific survival rates were 100%. In conclusion, an indolent long-term clinical course was confirmed in the patients with primary intestinal follicular lymphoma. The watch-and-wait strategy is a reasonable approach for the initial management of these patients.

Outcomes of Patients with Left-Sided Obstructive Colorectal Cancer: Comparison between Self-Expandable Metallic Stent and Other Treatment Methods.

INTRODUCTION: This multi-institutional retrospective study aimed to evaluate the efficacy of preoperative self-expandable metallic stent (SEMS) placement for patients with left-sided obstructive colorectal cancer (OCRC). METHODS: Overall, 520 consecutive patients who received treatment for OCRC were enrolled. Of these, the data of 253 patients who underwent primary tumour resection for left-sided OCRC were reviewed. The short- and long-term outcomes were compared between the SEMS group and other three groups: transanal decompression tube (TaDT), decompressing stoma (DS), and emergency resection (ER). RESULTS: The SEMS group had a higher frequency of laparoscopic surgery (p < 0.001), lesser frequency of postoperative stoma (p < 0.001), and more dissected lymph nodes (p < 0.001) than the other groups. Moreover, the SEMS group had shorter postoperative hospital stays than the TaDT, DS, and ER groups (p = 0.005, p = 0.037, and p < 0.001, respectively). The Kaplan-Meier survival curves of recurrence-free survival and overall survival did not differ significantly between the SEMS group and the other three groups in patients with stage II and III diseases. DISCUSSION/CONCLUSION: Elective surgery after SEMS placement may improve short-term outcomes compared to other treatment strategies, with similar long-term outcomes.

Surgical management and outcomes of obstructive colorectal cancer in elderly patients: A multi-institutional retrospective study.

BACKGROUND: The outcomes and prognostic factors of elderly patients with obstructive colorectal cancer are unknown. This was the focus of our multi-institutional retrospective study. METHODS: Medical records of 520 patients (elderly [group E, aged ≥75 years, n = 271]; and nonelderly [group NE, aged <75 years, n = 249]) who received treatment for obstructive colorectal cancer in 2008 to 2018 at 14 leading hospitals in Kagawa prefecture (Japan) were reviewed. Short- and long-term outcomes of patients who underwent tumor resection (n = 438) were compared between the groups. Their prognostic factors were identified. RESULTS: The tumor resection rate was 79% (n = 213) and 90% (n = 225) in groups E and NE, respectively. Group E had more emergency resections (27 [12.7%] vs 15 [6.6%], P = .037), shorter operating times (194 vs 221 min, P < .001), fewer dissected lymph nodes (14 vs 17, P = .004), and less adjuvant chemotherapy (47 [26.8%] vs 122 [76.7%], p < .001) than group NE. Postoperative complication rates and recurrence-free survival were not significantly different between the groups. Overall survival was significantly lower in group E than in group NE. Distant metastases and no postoperative chemotherapy were independent poor prognostic factors for overall survival in groups E and NE. Emergency resection (hazard ratio:1.83; 95% confidence interval: 1.02-3.26) was a significant poor prognostic indicator in group E only. CONCLUSIONS: The short-term outcomes and recurrence-free survival of elderly and nonelderly patients with obstructive colorectal cancer were similar, although the 90-day mortality rate of the elderly patients was higher. Furthermore, elective surgery after bowel decompression is associated with a better outcome in the elderly.

Dipeptide γ-secretase inhibitor treatment enhances the anti-tumor effects of cisplatin against gastric cancer by suppressing cancer stem cell properties.

The γ-secretase inhibitor blocks Notch activity by preventing its cleavage at the cell surface. In the present study, the effect of the γ-secretase inhibitor on the viability of gastric cancer cells when administered in combination with cisplatin was investigated, with particular focus on CD44highLgr-5high cancer cells. The four gastric cancer cell lines, MKN45, MKN74, SC-6-JCK and SH-10-TC, were used for the experiments. In the MTT assay, treatment with 25 µM dipeptide γ-secretase inhibitor (DAPT) alone did not affect cell proliferation in any of the four cell lines. Gastric cancer cells subjected to combination treatment with DAPT and cisplatin exhibited decreased viability when compared with those treated with cisplatin alone. Flow cytometry was performed to evaluate the expression of cluster of differentiation (CD)-44 and leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr-5), two cancer stem cell markers in gastric cancers. Treatment with cisplatin alone significantly increased the proportion of CD44highLgr-5high cells. However, the addition of DAPT to cisplatin reduced the CD44highLgr-5high fraction, suggesting that DAPT reduced the number of gastric cancer cells. In conclusion, the present study demonstrated the synergistic effects of DAPT in combination with cisplatin by decreasing the survival of gastric cancer cells. In addition, combination treatment with DAPT reduced the number of CD44highLgr-5high cells, which are thought to exhibit cancer stem cell properties. These results highlight the therapeutic potential of DAPT in gastric cancer treatment.

Promising therapeutic efficacy of a novel reduced expression in immortalized cells/dickkopf-3 expressing adenoviral vector for hepatocellular carcinoma.

BACKGROUND AND AIM: Reduced expression in immortalized cells (REIC)/dickkopf-3 (Dkk-3) is a tumor suppressor gene that is downregulated in various cancers. In our previous study of prostate cancer, the REIC/Dkk-3-expressing adenoviral vector (Ad-REIC) was found to induce cancer-selective apoptosis. This study recently developed a novel super gene expression (SGE) system and used this system to re-construct an Ad-REIC vector, termed the Ad-SGE-REIC, to achieve more effective therapeutic outcomes. In this study, the therapeutic effects of Ad-SGE-REIC on hepatocellular carcinoma (HCC) was assessed. METHODS: Human HCC cell lines (HLE, Huh7, HepG2, HLF, SK-Hep1, and PLC), human HCC tissues, and mouse HCC cell line (Hepa1-6) were used in this study. REIC/Dkk-3 expression was assessed by immunoblotting and immunohistochemistry. The relative cell viability and the apoptotic effect were examined in vitro, and the anti-tumor effects of Ad-SGE-REIC treatment were analyzed in the mouse xenograft model. This study additionally assessed anti-tumor immunological effects on the immunocompetent mice. RESULTS: REIC/Dkk-3 expression was decreased in HCC cell lines and HCC tissues. Ad-SGE-REIC reduced cell viability and induced apoptosis in HCC cell lines (HLE and Huh7), inhibited tumor growth in the mouse xenograft model, and demonstrated in vivo anti-cancer immunostimulatory effects on the HCC cell line (Hepa1-6). CONCLUSIONS: Ad-SGE-REIC treatment not only enhanced cell killing effects in vitro but also elicited significant therapeutic effects, with tumor growth suppression, in vivo. REIC/Dkk-3 gene therapy using Ad-SGE-REIC potentially represents an innovative new therapeutic tool for HCC.

[Corrigendum] Loss of Runt­related transcription factor 3 induces resistance to 5­fluorouracil and cisplatin in hepatocellular carcinoma.

Following the publication of this article, we realize that there were some errors in the manuscript. Details of the experiments describing the gene silencing of RUNX3 with small interfering RNA (siRNA) were erroneously included in this paper, and all references to siRNA should have been deleted from the manuscript prior to publication. In the subsection entitled 'Cell lines and cell culture' on page 2577, the left­hand column, the text should have indicated that the human HCC cell lines Hep3B and Huh7 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), whereas HLF cells were obtained from the Japanese Cancer Resources Bank (Tokyo, Japan). Lastly, an error was made in describing the calculation of the IC50 values, which did not correlate with the data shown in Fig. 2. Therefore, the subsection entitled 'Ectopic RUNX3 protein expression suppresses cell growth...' should have been entitled 'Ectopic RUNX3 protein expression increases 5­FU and CDDP sensitivity', and the text herein should have read as follows: We analyzed the effects of RUNX3 on chemosensitivity in the RUNX3­ or CAT (mock)­transfected Hep3B and Huh7 cells. RUNX3 expression enhanced 5­FU sensitivity in both cell lines; the cell viability with 5­FU (100 nM) decreased from 66.3±4.6 to 34.3±5.0%, and from 71.0±4.7% to 27.0±5.5% in the Hep3B and Huh7 cells, respectively (Fig. 2A). RUNX3 expression also enhanced CDDP sensitivity in both cell lines; the cell viability with CDDP (100 nM) decreased from 58.7±2.6% to 25.7±4.9%, and from 67.7±4.1% to 25.7±7.5% in the Hep3B and Huh7 cells, respectively (Fig. 2B). We sincerely apologize for these errors and oversights, which have not affected any of the overall conclusions reported in the study, and regret any inconvenience they may have caused. [the original article was published in the Oncology Reports 35: 2576-2582, 2016; DOI: 10.3892/or.2016.4681].

Loss of Runt-related transcription factor 3 induces resistance to 5-fluorouracil and cisplatin in hepatocellular carcinoma.

Runt-related transcription factor 3 (RUNX3) is known to function as a tumor suppressor in gastric cancer and other types of cancers, including hepatocellular carcinoma (HCC). However, its role has not been fully elucidated. In the present study, we aimed to evaluate the role of RUNX3 in HCC. We used the human HCC cell lines Hep3B, Huh7 and HLF; RUNX3 cDNA was introduced into Hep3B and Huh7 cells, which were negative for endogenous RUNX3 expression, and RUNX3 siRNA was transfected into HLF cells, which were positive for endogenous RUNX3. We analyzed the expression of RUNX3 and multidrug resistance-associated protein (MRP) by immunoblotting. MTT assays were used to determine the effects of RUNX3 expression on 5-fluorouracil (5-FU) and cisplatin (CDDP) sensitivity. Finally, 23 HCC specimens resected from patients with HCC at Okayama University Hospital were analyzed, and correlations among immunohistochemical expression of RUNX3 protein and MRP protein were evaluated in these specimens. Exogenous RUNX3 expression reduced the expression of MRP1, MRP2, MRP3 and MRP5 in the RUNX3-negative cells, whereas knockdown of RUNX3 in the HLF cells stimulated the expression of these MRPs. An inverse correlation between RUNX3 and MRP expression was observed in the HCC tissues. Importantly, loss of RUNX3 expression contributed to 5-FU and CDDP resistance by inducing MRP expression. These data have important implications in the study of chemotherapy resistance in HCC.

Synergistic anti-pancreatic cancer immunological effects by treatment with reduced expression in immortalized cells/dickkopf-3 protein and peripheral blood mononuclear cells.

BACKGROUND AND AIM: Reduced expression in immortalized cells/dickkopf-3 (REIC/DKK3) is a reported tumor suppressor gene and has potential to become an innovative therapy for various cancers. We examined the antitumor immunological effects of human REIC/DKK3 protein against pancreatic cancer. METHODS: Activation of extracellular signal-regulated kinases 1 and 2, mammalian target of rapamycin, and signal transducer and activator of transcription 3 by REIC/DKK3 protein was assessed in human peripheral blood mononuclear cells using immunoblotting. Pancreatic cancer cell lines (AsPC-1 and MIA Paca-2) were cocultured with peripheral blood mononuclear cells, and the anticancer effects of REIC/DKK3 protein were assessed using the methyl thiazole tetrazolium, cytotoxicity, and enzyme-linked immunospot assays. The antitumor immunological effects of the combined treatment with REIC/DKK3 protein and peripheral blood mononuclear cells were also assessed in a pancreatic cancer model using non-obese diabetic/severe combined immunodeficiency mice. RESULTS: The REIC/DKK3 protein activated extracellular signal-regulated kinases 1 and 2, mammalian target of rapamycin, and signal transducer and activator of transcription 3 in peripheral blood mononuclear cells. REIC/DKK3 protein inhibited in vitro cancer cell viability and enhanced cytotoxicity when incubated with peripheral blood mononuclear cells. REIC/DKK3 protein induced significant production of interferon gamma from lymphocytes incubated with pancreatic cancer cells, indicating that CD8+ T cells were activated in the peripheral blood mononuclear cells when cocultured with AsPC-1 and MIA Paca-2 in the presence of REIC/DKK3 protein. Combined treatment with REIC/DKK3 protein and peripheral blood mononuclear cells produced in vivo anticancer immunostimulatory effects on pancreatic cancer cells. CONCLUSIONS: The REIC/DKK3 protein and peripheral blood mononuclear cells synergistically enhanced anticancer immunological effects against pancreatic cancer cells. The observed immunomodulatory effect of combined treatment likely occurs in adenovirus-mediated REIC/DKK3 gene therapy and provides important clues to the therapeutic mechanisms involving immune cells.

Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells.

Microenvironment plays an important role in epithelial-mesenchymal transition (EMT) and stemness of cells in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) is known as a tumor stemness marker of HCC. To investigate the relationship between microenvironment and stemness, we performed an in vitro co-culture assay. Four HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) were co-cultured with the TWNT-1 immortalized hepatic stellate cells (HSCs), which create a microenvironment with HCC. Cell proliferation ability was analyzed by flow cytometry (FCM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while migration ability was assessed by a wound healing assay. Expression of EpCAM was analyzed by immunoblotting and FCM. HCC cell lines were co-cultured with TWNT-1 treated with small interfering RNA (siRNA) for TGF-ß and HB-EGF; we then analyzed proliferation, migration ability and protein expression using the methods described above. Proliferation ability was unchanged in HCC cell lines co-cultured with TWNT-1. Migration ability was increased in HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) directly (216.2±67.0, 61.0±22.0, 124.0±66.2 and 51.5±40.3%) and indirectly (102.5±22.0, 84.6±30.9, 86.1±25.7 and 73.9±29.7%) co-cultured with TWNT-1 compared with the HCC uni-culture. Immunoblot analysis revealed increased EpCAM expression in the HCC cell lines co-cultured with TWNT-1. Flow cytometry revealed that the population of E-cadherin-/N-cadherin+ and EpCAM-positive cells increased and accordingly, EMT and stemness in the HCC cell line were activated. These results were similar in the directly and indirectly co-cultured samples, indicating that humoral factors were at play. Conversely, HCC cell lines co-cultured with siRNA­treated TWNT-1 showed decreased migration ability, a decreased population of EpCAM-positive and E-cadherin-/N-cadherin+ cells. Taken together, humoral factors secreted from TWNT-1 promote upregulation of EpCAM and EMT in hepatic cancer cells.

Potential of adenovirus-mediated REIC/Dkk-3 gene therapy for use in the treatment of pancreatic cancer.

BACKGROUND AND AIM: The reduced expression in immortalized cells REIC/the dickkopf 3 (Dkk-3) gene, tumor suppressor gene, is downregulated in various malignant tumors. In a prostate cancer study, an adenovirus vector carrying the REIC/Dkk-3 gene (Ad-REIC) induces apoptosis. In the current study, we examined the effects of REIC/Dkk-3 gene therapy in pancreatic cancer. METHODS: REIC/Dkk-3 expression was assessed by immunoblotting and immunohistochemistry in the pancreatic cancer cell lines (ASPC1, MIAPaCa2, Panc1, BxPC3, SUIT-2, KLM1, and T3M4) and pancreatic cancer tissues. The Ad-REIC agent was used to investigate the apoptotic effect in vitro and antitumor effects in vivo. We also assessed the therapeutic effects of Ad-REIC therapy with gemcitabine. RESULTS: The REIC/Dkk-3 expression was lost in the pancreatic cancer cell lines and decreased in pancreatic cancer tissues. Ad-REIC induced apoptosis and inhibited cell growth in the ASPC1 and MIAPaCa2 lines in vitro, and Ad-REIC inhibited tumor growth in the mouse xenograft model using ASPC1 cells. The antitumor effect was further enhanced in combination with gemcitabine. This synergistic effect may be caused by the suppression of autophagy via the enhancement of mammalian target of rapamycin signaling. CONCLUSIONS: Ad-REIC induces apoptosis and inhibits tumor growth in pancreatic cancer cell lines. REIC/Dkk-3 gene therapy is an attractive therapeutic tool for pancreatic cancer.

Loss of runt-related transcription factor 3 induces gemcitabine resistance in pancreatic cancer.

BACKGROUND & AIM: Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene that is expressed in gastric and other cancers including pancreatic cancer. However, the precise function of RUNX3 in pancreatic cancer has not been fully elucidated. In this study, we aimed to determine the effect of decreased RUNX3 expression in pancreatic cancer. METHODS: This study included 36 patients with primary pancreatic cancer, who had undergone pancreaticoduodenectomy. All patients were treated with 1000 mg/m2 gemcitabine after the surgery. The pancreatic cancer cell lines PANC-1, MIAPaCa-2, BxPC-3, SUIT-2, and KLM-1 were used for immunoblotting analysis of RUNX3 and multidrug resistance protein (MRP) expressions. Ectopic RUNX3 expression was achieved by cDNA transfection of the cells, and small interfering RNA (siRNA) against RUNX3 was used to knock down endogenous RUNX3. Cell growth in the presence of gemcitabine was assessed using the MTT assay. RESULTS: Patients with RUNX3-positive and RUNX3-negative pancreatic cancer had a median survival of 1006 and 643 days, respectively. Exogenous RUNX3 expression reduced the expression of MRP1, MRP2, and MRP5 in endogenous RUNX3-negative cells, whereas RUNX3 siRNA increased the expressions of these genes in endogenous RUNX3-positive cells. Exogenous RUNX3 expression decreased gemcitabine IC50 in RUNX3-negative cells. CONCLUSION: Loss of RUNX3 expression contributes to gemcitabine resistance by inducing MRP expression, thereby resulting in poor patient survival.

Duodenal duplication cyst of the ampulla of Vater.

A 35-year-old man presented with the complaint of epigastric discomfort. Gastrointestinal endoscopy and endoscopic ultrasonography revealed a cystic lesion 20 mm in size at the ampulla of Vater. Endoscopic retrograde cholangiopancreatography (ERCP) revealed that the cystic lesion communicated with both the common bile duct and pancreatic duct via the common channel. Choledochocele was ruled out by close examination of the ERCP findings. The cystic lesion was surgically resected. Since histological findings revealed that the mucosa inside the lesion was duodenum-like and contained a layer of smooth muscle, the lesion was diagnosed as a congenital duplication cyst of the duodenum.