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Background Immune checkpoint inhibitors targeting the programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway have demonstrated antitumor effects in patients with various malignancies, including esophageal cancer. Thus, a better understanding of local immunity in esophageal cancer is crucial for improving treatment and clinical outcomes. Methods We evaluated PD-1 expression on tumor-infiltrating lymphocytes (TILs), as well as PD-L1 expression on cancer cells, by immunohistochemistry and immunofluorescence using a nonbiased database of 433 curatively resected esophageal cancers. With the idea of application as liquid biopsy, PD-1 expression status on peripheral lymphocytes was evaluated by flow cytometry. Results The cutoff value of PD-1 expression was the median PD-1 count. Compared with cases of low PD-1 expression (n = 219), cases with high levels of PD-1 expression (n = 213) showed significantly worse overall survival (log-rank P = .0017). The prognostic effect of PD-1 differed according to the preoperative treatment status (P for interaction = .040); PD-1 expression was associated with high overall mortality among patients without preoperative therapy, while no such association was present among those with preoperative treatment. A stratification based on PD-1 and PD-L1 status was also significantly associated with overall survival (log-rank P = .0005). PD-1 expression on TILs was significantly associated with that on peripheral lymphocytes (P Conclusions PD-1 expression on TILs was associated with an unfavorable clinical outcome in esophageal cancer, supporting its role as a prognostic biomarker. The combination of PD-1 and PD-L1 expression enabled further classification of patients according to clinical outcome.

Experimental evidence suggests a role of intratumour Fusobacterium nucleatum in the aggressive behaviour of gastrointestinal cancer through downregulating anti-tumour immunity. We investigated the relationship between intratumour F. nucleatum and immune response to oesophageal cancer.Utilising an unbiased database of 300 resected oesophageal cancers, we measured F. nucleatum DNA in tumour tissue using a quantitative polymerase chain reaction assay, and evaluated the relationship between the abundance of F. nucleatum and the densities of T cells (CD8 + , FOXP3 + and PDCD1 + ), as well as lymphocytic reaction patterns (follicle lymphocytic reaction, peritumoural lymphocytic reaction, stromal lymphocytic reaction and tumour-infiltrating lymphocytes) in oesophageal carcinoma tissue.F. nucleatum was significantly and inversely associated only with the peritumoural lymphocytic reaction (P = 0.0002). Compared with the F. nucleatum-absent group, the F. nucleatum-high group showed a much lower level of the peritumoural lymphocytic reaction (univariable odds ratio, 0.33; 95% confidence interval, 0.16-0.65; P = 0.0004). A multivariable model yielded a similar finding (multivariable odds ratio, 0.34; 95% confidence interval 0.16-0.69; P = 0.002).Intratumour F. nucleatum is associated with a diminished peritumoural lymphocytic reaction, providing a platform for further investigations on the potential interactive roles between intratumour F. nucleatum and host immunity.

Background The incidence of synchronous gastrointestinal neuroendocrine tumors (GI-NETs) and colorectal cancer is very low. Case presentation We present a 72-year-old man diagnosed with a rectal neuroendocrine tumor (NET) with multiple organ metastases and simultaneous sigmoid colon cancer. Although the NET was his prognostic factor, he underwent a laparoscopic sigmoidectomy at first because it was expected that the colon cancer would cause obstruction or bleeding during NET treatment. Subsequently, he started taking everolimus. Conclusions We should consider surgical resection of the synchronous cancer before systemic therapy for a GI-NET regardless of the difference in prognosis between synchronous tumors, if the cancer may impair the continuation of systemic therapy.

Indoleamine 2, 3‐dioxygenase 1 (IDO1) is a primary enzyme that generates immunosuppressive metabolites. It plays a major role in tumor immunology and is a potential immune‐based therapeutic target. We have reported that IDO1 protein expression was associated with an unfavorable clinical outcome in esophageal cancer. Recently, it has been reported that IDO1 expression is regulated by methylation of the IDO1 promoter. Thus, the aim of this study was to examine the relationship between IDO1 expression, IDO1 promoter methylation, and clinicopathological features in esophageal cancer. We first confirmed changes in IDO1 expression levels in vitro by treating cells with 5‐azacytidine. We then evaluated the relationship between IDO1 expression levels, IDO1 promoter methylation (bisulfite pyrosequencing), and clinicopathological features using 40 frozen samples and 242 formalin‐fixed, paraffin‐embedded samples resected from esophageal cancer patients. We treated cell lines with 5‐azacytidine, and the resulting hypomethylation induced significantly higher IDO1 expression (P

Introduction Tumour-associated macrophages (TAMs) in tumour microenvironments promote cancer cell proliferation, immunosuppression and angiogenesis, leading to tumour growth and metastasis. TAMs have become increasingly recognised as a cancer therapy target, such as in combination therapy with an immunity checkpoint inhibitor. However, the clinical and prognostic features of TAMs, and the relationship between TAMs and programmed death ligand 1 (PD-L1), remain unexplored in oesophageal cancer. Methods Using a non-biased database of 305 resected oesophageal cancer preparations, we evaluated the expression of two M2-like macrophage markers (CD163 and CD204) and PD-L1 on tumour cells by immunostaining. Through in vitro assays, we examined how TAMs influence phenotypic malignancy and PD-L1 expression. Results High density of CD163 (n = 160) or CD204 (n = 146) was associated with significantly worse overall survival than low expression (log rank P = 0.0025 and 0.018 for CD163 and CD204, respectively). The prognostic effect of TAMs was not significantly modified by any clinical factors (P > 0.05 for all interactions). High TAM density was significantly associated with increased PD-L1 expression. In in vitro assays, cell invasion and migration ability were significantly more upregulated in oesophageal cancer cell lines cocultured with activated macrophages than in control cell lines. Coculture with activated macrophages elevated the PD-L1 expression in cancer cells. Conclusions High TAM density in oesophageal cancer tissues was associated with shorter survival, suggesting a prognostic biomarker role for TAMs. TAMs also increase PD-L1 expression in tumour cells. Given the significant interest in cancer immunotherapies targeting TAMs and PD-L1, the current findings should have considerable clinical implications.

The effect of resection of the thoracic duct (TD) along with surrounding lymph nodes (LN) on short- and long-term outcomes of esophagectomy in esophageal cancer patients is not well defined. A total of 537 consecutive patients suffering from esophageal cancer who underwent three-incision esophagectomy between April 2005 and August 2018 were eligible for short-term outcome analysis. Among them, 487 patients who underwent surgery before August 2017 were eligible for analysis of long-term outcomes. Moreover, 164 patients who underwent esophagectomy after August 2012 and had no recurrence at 1-year postoperative follow-up were prospectively investigated for postoperative nutritional status. A total of 145 patients (27.0%) underwent TD resection with surrounding LN. Since the clinical stage was significantly more advanced in the removal group, preoperative treatment was more frequently performed in them. The operative time was significantly longer in the removal group. Intraoperative bleeding was higher in the removal group. Morbidity of Clavien–Dindo classification (CDc) ≥ II and pulmonary morbidities were frequently observed in the removal group. Multivariate analysis suggested that TD resection was an independent risk factor for pulmonary morbidities. Moreover, it may be associated with the incidence of CDc ≥ II morbidity. Greater numbers of LN were dissected in the thorax of patients in the removal group. However, overall survival was equivalent irrespective of the TD procedure in each stage. Nutritional status at 1-year follow-up was equivalent between the groups. On the basis of the present results, routine removal of the TD during esophagectomy is not recommended.

Background: Regorafenib and TAS-102 are standard salvage-line treatment options for patients with chemorefractory metastatic colorectal cancer (mCRC). We aimed to evaluate the prognostic significance of skeletal muscle mass in mCRC patients receiving these salvage-line therapies. Methods: We conducted a retrospective analysis of 52 patients with mCRC who received regorafenib or TAS-102 as salvage-line treatment. Skeletal muscle cross-sectional area was measured by pretreatment CT to obtain the skeletal muscle index (SMI, cm2/m2). We divided patients into 2 groups (low-SM/high-SMI) based on the median value of SMI. Result: The median SMI was 51.8 cm2/m2 in males and 39.2 cm2/m2 in females. In Kaplan-Meier analysis, patients in the low-SMI group showed significantly shorter overall survival (3.7 vs. 7.3 months, log-rank p = 0.002) and progression-free survival (1.9 vs. 2.8 months, log-rank test p = 0.009) than those in the high-SMI group. Subsequent multivariate analysis revealed that the SMI was an independent prognostic factor (hazard ratio = 2.381, 95% CI 1.189–4.944, p = 0.014). Patients in the low-SMI group had significantly more grade 3 or 4 adverse events than those in the high-SMI group (46 vs.12%, p = 0.013). Conclusion: Low skeletal muscle mass is a negative factor for survival outcomes in mCRC patients treated with salvage-line chemotherapy.

Iron deficiency anemia is represented in colorectal cancer (CRC) patients. Iron surplus load to increase non-transferrin bound iron (NTBI), and NTBI promotes cancer progression and influences microbiota. This study investigated whether preoperative serum iron status was associated with prognosis after CRC resection.We evaluated preoperative iron and transferrin saturation (TSAT), which was calculated as iron divided by total iron-binding capacity, in 327 patients who underwent surgery for Stage II-III CRC. Fe 60 μg/dl and TSAT 40% were defined as low and high iron, respectively. The associations between iron status and overall survival (OS) were evaluated in univariate and multivariate Cox proportional hazards analysis.Of the 327 patients, 179 (54.7%), 124 (37.9%) and 24 (7.3%) had low, normal and high iron, respectively. In univariate analysis, low iron was associated with shorter OS (hazard ratio [HR] 2.821, 95% confidence interval [CI] 1.451-5.485, P = 0.002). High iron was also associated with shorter OS (HR 3.396, 95% CI 1.359-8.489, P = 0.009). In multivariate analysis, high age (P = 0.002), depth of invasion pT4 (P = 0.012), lymph-node metastasis presence (P = 0.035), low albumin (P = 0.011), low iron (HR 2.282, 95% CI 1.163-4.478, P = 0.016) and high iron (HR 3.757, 95% CI 1.486-9.494 P = 0.005) were independently associated with shorter OS. High iron was associated with the amount of intratumoral Fusobacterium nucleatum compared with normal iron.Both low and high preoperative iron in Stage II-III CRC patients were associated with unfavorable OS in univariate and multivariate analyses.

Liver fibrosis influences liver regeneration and surgical outcomes, and several noninvasive models based on laboratory data have been developed to predict liver fibrosis. This study was performed to determine whether the Fibrosis-4 (FIB-4) index, a noninvasive fibrosis marker, can predict the prognosis in patients with colorectal liver metastases (CRLM) undergoing hepatectomy. This retrospective study involved 193 consecutive patients with CRLM who underwent hepatectomy. The FIB-4 index was calculated by laboratory data and age before hepatectomy and before preoperative chemotherapy. The FIB-4 cut-off was determined using survival classification and regression tree analysis. Patients were divided into two groups (high and low FIB-4 index), and post-hepatectomy overall survival (OS) and recurrence-free survival (RFS) were investigated. In total, 193 patients were evaluated. Chemotherapy before hepatectomy was performed in 105 (54.4%) patients. A high FIB-4 index (> 2.736) was found in 39 (20.2%) patients. OS was significantly shorter in patients with a high FIB-4 index than those with a low FIB-4 index in the univariate (45.9 vs. 74.4 months, log-rank p = 0.007) and multivariate analysis (hazard ratio 2.28, 95% confidence interval 1.39–3.74; p = 0.001). Among patients who received chemotherapy before hepatectomy, those with a high FIB-4 index had significantly shorter RFS (6.9 vs. 45.3 months, log-rank p = 0.047) and OS (23.9 vs. 55.0 months, log-rank p = 0.003) than those with a low FIB-4 index. This association was also confirmed by multivariate analysis (hazard ratio 4.28, 95% confidence interval 1.46–12.6; p = 0.008). Both the preoperative and prechemotherapy FIB-4 index can predict long-term outcomes after hepatectomy in patients with CRLM.

Radiofrequency ablation (RFA) can be a minimally invasive therapeutic option in patients with lung metastasis from colorectal caner. We aimed to elucidate the safety and survival benefit of computed tomography (CT)-guided percutaneous RFA for lung metastasis from colorectal cancer. A total 188 lesions were ablated in 43 patients from 2005 to 2017. The clinicopathological and survival data of patients were collected retrospectively. The short- and long-term outcomes and prognostic factors were analyzed. Eight patients (18.6%) had viable extrapulmonary metastasis at RFA treatment. The median number of treated lung tumors was 2, and the median maximum diameter was 12 mm. Complications, such as pneumothorax, pleural effusion and subcutaneous emphysema, occurred in 24 (55.8%) patients. Although chest tube drainage for pneumothorax was needed in 6 patients (14.0%), there were no mortalities. Repeated RFA for lung recurrence after primary RFA was performed in 14 patients (32.6%). In a median follow-up of 24.3 months, the median progression-free and overall survival (OS) were 6.8 months and 52.7 months, respectively. The presence of extrapulmonary metastasis and a maximum tumors size of > 15 mm were independently associated with a worse disease-free survival and OS. The OS of patients who underwent repeated RFA was significantly better than that of patients who underwent RFA only once. CT-guided percutaneous RFA for lung metastasis from colorectal cancer is a safe and effective procedure in patients not eligible for surgery, particularly for lesions smaller than 1.5 cm without extrapulmonary metastasis.

Peritoneal dissemination is the most common metastatic pattern in advanced gastric cancer (GC) and has a very poor prognosis. However, its molecular mechanism has not been elucidated. Our study investigated genes associated with peritoneal dissemination of GC. We performed combined expression analysis of metastatic GC cell lines and identified Procollagen-lysine, 2-oxoglutarate 5-dioxygenase2 (PLOD2) as a potential regulator of peritoneal dissemination. PLOD2 is regulated by hypoxia-inducible factor-1 (HIF-1) and mediates extracellular matrix remodeling, alignment, and mechanical properties. We analyzed PLOD2 expression immunohistochemically in 179 clinical samples, and found high PLOD2 expression to be significantly associated with peritoneal dissemination, leading to poor prognosis. In an in vivo-collected metastatic cell line, downregulation of PLOD2 by siRNA reduced invasiveness and migration. Hypoxia upregulated PLOD2 mediated by HIF-1, and promoted invasiveness and migration. After exposure to hypoxia, a cell line transfected with siPLOD2 exhibited significantly suppressed invasiveness and migration, despite high HIF-1 expression. These findings indicate that PLOD2 is a regulator of, and candidate therapeutic target for peritoneal dissemination of GC. Although peritoneal dissemination of GC has a very poor prognosis, its molecular mechanism has not been elucidated. We identified PLOD2 regulated by HIF-1 as a potential regulator of peritoneal dissemination of GC. Finally, we showed that PLOD2 promotes cell invasiveness and migration in GC under hypoxia and lead to peritoneal dissemination of GC.

Patients with gastric cancer (GC) are affected by changes in iron status. Before surgery, GC patients are likely to have iron-deficiency anemia; and after gastrectomy, patients suffer from low nutritional status and low iron. This study investigated preoperative iron status associated with prognosis after curative gastrectomy for gastric cancer. We evaluated preoperative serum hemoglobin (Hgb), Fe and total iron-binding capacity (TIBC) in 298 patients who underwent curative gastrectomy for GC without preoperative chemotherapy, and analyzed these factors’ associations with prognosis after surgery. Of the 298 patients, 129 (43.2%) had low Hgb levels, and 33 (11.1%) had low TIBC (

Programmed death ligand 1 (PD-L1) expression as a predictive biomarker for programmed cell death 1 (PD-1) inhibitor efficacy in gastric cancer (GC) remains controversial. We hypothesised that the conflicting results may be due to the inaccurate assessment of PD-L1 expression using biopsy samples. A total of 191 patients with GC who received radical resection were enrolled. PD-L1 expressions in biopsy and paired resected samples by immunohistochemistry staining were compared according to the number of biopsies. The numbers of PD-L1-positive patients determined by biopsy and resected samples were 89 (46.6%) and 135 (70.1%), respectively. The accordance rate was 64.4% (κ = 0.31). Single biopsy showed a lower accordance rate compared with multiple biopsies. Our study revealed that single biopsy cannot fully reflect PD-L1 expression in the whole tumour in GC. Multiple biopsies are recommended for accurate diagnosis of PD-L1 expression in GC.

The neutrophil-to-lymphocyte ratio (NLR) has been reported to predict the prognosis of various malignant tumors, including esophageal cancer. However, no previous reports have supported the use of the preoperative NLR as an independent prognostic marker focused on superficial (T1) esophageal cancer. The aim of this study was to elucidate the prognostic impact of the preoperative NLR in T1 esophageal cancer. This retrospective study recruited 245 consecutive patients with T1 esophageal cancer who underwent subtotal esophagectomy between 2005 and 2016. The relationship between the preoperative NLR and clinicopathological characteristics was analyzed. The preoperative NLR was significantly higher in male patients (p = 0.029), patients with T1b esophageal cancer (p = 0.0274), and patients with venous vessel invasion (p = 0.0082). In the Kaplan–Meier analysis, the elevated preoperative NLR was significantly associated with a poorer disease-free survival (p

The aim of this study was to elucidate the risk factors for and prognostic value of lateral pelvic lymph node (LPLN) metastasis in advanced rectal cancer patients, including those with stage IV disease. The treatment outcomes of 78 patients with advanced rectal cancer, the lower margin of which was located at or below the peritoneal reflection, who underwent curative-intent surgery with bilateral LPLN dissection from 2005 to 2018 were retrospectively analyzed. In total, 78 rectal cancer patients, including 13 patients with stage IV tumors, 9 patients (11.5%) had LPLN metastasis. A multivariate analysis to identify preoperative clinical factors associated with LPLN metastasis showed that tumor location (below the peritoneal reflection: Rb), LPLN metastasis on preoperative imaging and distant metastasis were independent predictors of LPLN metastasis. In addition, metastasis at the regional lymph nodes in the mesorectum was significantly associated with LPLN metastasis. Both the disease-free survival (DFS) and cancer-specific survival (CSS) of patients with LPLN metastasis were significantly worse in comparison to patients without LPLN metastasis, and the CSS of stage IV patients with LPLN metastasis was significantly worse in comparison to stage IV patients without LPLN metastasis. Tumor location (Rb), LPLN metastasis on preoperative imaging and distant metastasis were risk factors for LPLN metastasis. The prognosis of rectal cancer patients with LPLN metastasis is poor. There may not be the indication of LPLN dissection in stage IV lower rectal cancer except cases having complaints due to LPLN metastasis.

Respiratory morbidity is common after esophagectomy and can be a major cause of surgery-related mortality. Thus, it is important to identify novel predictors that can preoperatively estimate the incidence of postoperative respiratory morbidity. Asymptomatic sputum in the respiratory tract is sometimes observed on preoperative computed tomography (CT). This study aimed to determine the clinical importance of sputum in the respiratory tract as a predictor of postoperative morbidity after esophagectomy for esophageal cancer. The study included 609 consecutive patients who underwent three-incisional esophagectomy for esophageal cancer between April 2005 and November 2018. Among the patients, 76 (12.5%) had sputum in the respiratory tract on preoperative CT. This finding was significantly associated with older age, more extreme smoking habit, worse performance status, lower forced expiratory volume 1%, and more frequent pulmonary comorbidities. Additionally, the incidence of postoperative pneumonia was higher in these patients than in those without sputum (16 vs 8%, p = 0.028). Sputum in the main bronchus was associated with higher frequencies of morbidity of Clavien–Dindo classification (CDc) ≥ II (p = 0.019), severe morbidity of CDc ≥ IIIb (p = 0.058), pneumonia (p = 0.10), and pulmonary morbidity (p = 0.19) compared with the finding of sputum in the trachea alone. On multivariate analysis, sputum in the respiratory tract was an independent risk factor (hazard ratio, 2.07; 95% confidence interval, 1.019–4.207; p = 0.044) for postoperative pneumonia. Sputum in the respiratory tract is a novel predictor of postesophagectomy pneumonia. Patients with sputum in the more distal respiratory tract might have high risk of postoperative morbidities.

Few reliable prognostic markers have been established despite elucidation of the molecular mechanisms of gastrointestinal stromal tumor (GIST) development. We evaluated F-box and WD repeat domain-containing 7 (FBXW7), a cell-cycle-regulating and tumor suppressor, in GISTs. We aimed to determine the clinical relevance of FBXW7 in GISTs and characterize the molecular mechanism of FBXW7 in a GIST cell line. We measured FBXW7 expression in 182 GIST cases, correlated the expression levels with clinicopathological features, and characterized the molecular mechanism underlying suppressed FBXW7 expression in GIST cells in vitro. Of the 182 GISTs, 98 (53.8%) and 84 (46.2%) were categorized in the high and low FBXW7 expression groups, respectively. Compared with the high FBXW7 expression group, the low expression group showed a significantly poorer prognosis in terms of recurrence-free (P = 0.01) and overall (P = 0.03) survival. FBXW7 expression was a significant independent factor affecting the 10-year recurrence-free survival rate (P = 0.04). In vitro, FBXW7-specific siRNAs enhanced c-myc and Notch 1 protein expression and upregulated cell proliferation, invasion, and migration. FBXW7 is a potential predictive marker of recurrence after curative resection of GISTs. FBXW7 expression may help identify patients benefitting from adjuvant therapy more precisely compared with a conventional risk stratification model.

To determine whether prognostic nutritional index (PNI) affects clinical outcome through local immunity in esophageal cancers.PNI is an indicator of nutritional status and systemic immune competence, and has attracted attention as a prognostic biomarker. Tumor-infiltrating lymphocytes (TILs) are a specific histological feature of human cancers, reflecting an individual's immunological tumor response.Using a nonbiased database of 337 curatively resected esophageal cancers, we evaluated the relationship between PNI, TILs status, CD8 expression by immunohistochemical staining, and clinical outcome.Compared with PNI-high cases (n = 220), PNI-low cases (n = 117) showed significantly worse overall survival (log-rank P0.001; hazard ratio: 2.23; 95% confidence interval: 1.56-3.18; P0.001; multivariate hazard ratio: 1.67; 95% confidence interval: 1.14-2.44; P = 0.008). The TILs status was also significantly correlated with overall survival (P0.001). In addition, PNI was significantly associated with TILs status (P0.001) and the CD8-positive cell count (P = 0.041). A significant relationship between the peripheral blood lymphocyte count and TILs status was also observed (P0.001).PNI and TILs score expression were associated with clinical outcome in esophageal cancer, supporting their roles as prognostic biomarkers. Considering the relationship between PNI and TILs, nutritional status and systemic immune competence may influence patient prognosis through local immune response.

As cancer advances, changes in the systemic inflammatory response alter the relative levels of circulating white blood cell types and may contribute to the progression and outcomes of cancer. The aim of the current study is to clarify the impact of the preoperative neutrophil/lymphocyte ratio (NLR) on the prognosis and whether clinical and pathological features modify the influence of the NLR on the prognosis of esophageal squamous cell carcinoma (ESCC) patients. We retrospectively analyzed the mortality hazard ratios (HRs), including the preoperative NLR, obtained from data for 283 ESCC patients undergoing resection in the period from 2005 to 2011, adjusting for clinical and pathological features. A high NLR was associated with a significantly shorter overall survival (p = 0.0018) and cancer-specific survival (p = 0.0097). In the multivariate Cox model, we confirmed that the NLR was an independent prognostic marker for both overall survival (p = 0.0081) and cancer-specific survival (p = 0.028). The outcomes were not significantly modified by other diagnostic factors, including the tumor stage, in the survival analysis. The preoperative NLR is significantly associated with a poor prognosis in patients with ESCC, suggesting the utility of NLR as a cost-effective and broadly available independent prognostic marker of ESCC.

The adenoma-carcinoma sequence, the sequential mutation and deletion of various genes by which colorectal cancer progresses, is a well-established and accepted concept of colorectal cancer carcinogenesis. Proteins of the polycomb repressive complex 2 (PRC2) function as transcriptional repressors by trimethylating histone H3 at lysine 27; the activity of this complex is essential for cell proliferation and differentiation. The histone methyltransferase enhancer of zeste homolog 2 (EZH2), an essential component of PRC2, is associated with the transcriptional repression of tumor suppressor genes. EZH2 expression has previously been reported to increase with the progression of pancreatic intraductal papillary mucinous neoplasm. Thus, we hypothesized that EZH2 expression also increases during the adenoma-carcinoma sequence of colorectal cancer. The present study investigated changes in EZH2 expression during the colorectal adenoma-carcinoma sequence. A total of 47 patients with colorectal adenoma, 20 patients with carcinoma in adenoma and 43 patients with colorectal carcinoma who underwent surgical or endoscopic resection were enrolled in this study. Non-cancerous tissue from the clinical specimens was also examined. The association between EZH2 expression, pathology and expression of tumor suppressor genes during colorectal carcinogenesis were analyzed. Each specimen was immunohistochemically stained for EZH2, proliferation marker protein Ki-67 (Ki-67), cyclin-dependent kinase inhibitor (CDKN) 1A (p21), CDKN1B (p27) and CDKN2A (p16). Total RNA was extracted from formalin-fixed paraffin-embedded blocks and reverse transcription-quantitative polymerase chain reaction analysis of these genes was performed. Ki-67 and EZH2 expression scores increased significantly during the progression of normal mucosa to adenoma and carcinoma (P=0.009), and EZH2 expression score was positively associated with Ki-67 expression score (P=0.02). Conversely, p21 mRNA and protein expression decreased significantly, whereas expression of p27 and p16 did not change significantly. During the carcinogenesis sequence from normal mucosa to adenoma and carcinoma, EZH2 expression increased and p21 expression decreased significantly. EZH2 may therefore contribute to the development of colorectal cancer from adenoma via suppression of p21.

Background: Immunotherapies targeting the PD-1/PD-L1 pathway have emerged as promising therapeutic strategies for many cancers. PD-L1 and PD-L2 are ligands of PD-1 on T-cells and prevent T-cells activation and proliferation. There have been reported that PD-L1 and PD-L2 are expressed on not only antigen-presenting cells but also some types of tumors. Clinical and prognostic features of the PD-L1 have been widely reported for many types of cancers but those of PD-L2 remain unclear in esophageal cancer. The aim of this study was to analyze the clinical significance and the difference between PD-L1 and PD-L2 in esophageal cancer. Methods: Using a database of 437 curatively resected esophageal cancer from April 2005 to March 2016, we evaluated PD-L1 and PD-L2 expression in tumor cells by immunohistochemical staining and analyzed the association between those with clinicopathological features. Double immunohistochemical staining for PD-L1 and PD-L2 was performed to confirm the localization of each positive cell. Results: Out of 437 cases of esophageal cancer, 69 cases (15.8%) showed PD-L1 positive and 71 cases (16.2%) showed PD-L2 positive expression. There was no significant relationship between PD-L1 and PD-L2 positive cases (p=0.18). In both PD-L1 and PD-L2 positive cases, double immunohistochemical staining for PD-L1 and PD-L2 showed that there was a heterogeneity for PD-L1 and PD-L2 positive tumor cell. PD-L1 positive cases were significantly associated with tobacco use (p=0.013), pathological stage (p=0.001), and preoperative therapy present cases (p=0.015). In the other hand, PD-L2 positive cases were significantly associated with younger age (p=0.029). Thus PD-L1 and PD-L2 patient characteristics were likely different. Overall survival rates of the patients with PD-L1 or PD-L2 positive were significantly worse than those with PD-L1 or PD-L2 negative (p=0.009 and p=0.013, respectively). Conclusion: There was no significant relationship between PD-L1 and PD-L2 expression, and patient characteristics of those were different in patients with esophageal cancer. But both PD-L1 and PD-L2 expression were significantly associated with prognosis, supporting their roles as prognostic biomarkers. Citation Format: Kazuo Okadome, Yoshifumi Baba, Taisuke Yagi, Yuki Kiyozumi, Kojiro Eto, Yukiharu Hiyoshi, Yohei Nagai, Takatsugu Ishimoto, Masaaki Iwatsuki, Shiro Iwagami, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Clinical significance and the difference between programmed death-1 ligand-1 and ligand-2 in patients with esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4123.

Insulinomas are the most common functioning pancreatic neuroendocrine tumors (PNETs). We herein present the case of a 5-year survivor with insulinoma after complete response of postoperative liver metastases to hepatic arterial infusion combined with systemic chemotherapy. A 58-year-old woman was admitted to our hospital following loss of consciousness. Examination revealed a pancreatic tumor, and she underwent distal pancreatectomy following diagnosis of insulinoma. Superparamagnetic iron oxide magnetic resonance imaging (SPIO-MRI) revealed multiple liver metastases 3 months after surgery. Therefore, we performed hepatic arterial infusion of 5-fluorouracil (5-FU) combined with systemic gemcitabine infusion. We observed complete ablation of all metastatic liver nodules after 11 cycles of the chemotherapy using MRI. We continued this chemotherapy regimen for 20 cycles, and the patient remains alive without any recurrence 7 years after surgery.

Trastuzumab in combination with cisplatin and capecitabine could be a new standard treatment for patients with HER2-positive advanced gastric cancer. We herein report a case of advanced gastric cancer with peritoneal dissemination that was successfully treated with trastuzumab combined with cisplatin and capecitabine followed by gastrectomy. A 49-year-old man complained of epigastric pain and was diagnosed with advanced gastric cancer. A staging laparoscopy revealed peritoneal dissemination that was overexpressing HER2 protein. After five courses of chemotherapy comprising trastuzumab, cisplatin and capecitabine, laparoscopic restaging was performed. The disseminated nodules had disappeared, and biopsy of the scar of peritoneal seeding and cytology of the peritoneal lavage fluid both found no malignant cells. Therefore, total gastrectomy with D2 lymph node dissection was performed. Pathologically, there were no residual tumor cells in the resected stomach or peritoneal wall. Trastuzumab in combination with chemotherapy may have curative potential for peritoneal dissemination from HER2-positive gastric cancer.

Pneumonia is a major cause of postesophagectomy mortality and worsens the long-term survival in resected esophageal cancer patients. Moreover, preoperative treatments such as chemotherapy or chemoradiotherapy (which have recently been applied worldwide) might affect the bacterial flora of the sputum. To investigate the association among preoperative treatments, the bacterial flora of sputum, and the clinical and pathological features in resected esophageal cancer patients, this study newly investigates the effect of preoperative treatments on the bacterial flora of sputum. We investigated the association among preoperative treatments, the bacterial flora of sputum, and clinical and pathological features in 163 resected esophageal cancer patients within a single institution. Pathogenic bacteria such as Candida (14.1%), Staphylococcus aureus (6.7%), Enterobacter cloacae (6.1%), Haemophilus parainfluenzae (4.9%), Klebisiella pneumoniae (3.7%), Methicillin-resistant Staphylococcus aureus (MRSA) (3.7%), Pseudomonas aeruginosa (2.5%), Escherichia coli (1.8%), Streptococcus pneumoniae (1.8%), and Haemophilus influenzae (1.2%) were found in the sputum. The pathogen detection rate in the present study was 34.3% (56/163). In patients with preoperative chemotherapy and chemoradiotherapy, the indigenous Neisseria and Streptococcus species were significantly decreased (P= 0.04 and P= 0.04). However, the detection rates of pathogenic bacteria were not associated with preoperative treatments (all P> 0.07). There was not a significant difference of hospital stay between the sputum-monitored patients and unmonitored patients (35.5 vs. 49.9 days; P= 0.08). Patients undergoing preoperative treatments exhibited a significant decrease of indigenous bacteria, indicating that the treatment altered the bacterial flora of their sputum. This finding needs to be confirmed in large-scale independent studies or well-designed multicenter studies.

// Kenichi Nakamura 1 , Yoshifumi Baba 1 , Keisuke Kosumi 1 , Kazuto Harada 1 , Hironobu Shigaki 1 , Keisuke Miyake 1 , Yuki Kiyozumi 1 , Mayuko Ohuchi 1 , Junji Kurashige 1 , Takatsugu Ishimoto 1 , Masaaki Iwatsuki 1 , Yasuo Sakamoto 1 , Naoya Yoshida 1 , Masayuki Watanabe 2 , Mitsuyoshi Nakao 3 , Hideo Baba 1 1 Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan 2 Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan 3 Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan Correspondence to: Hideo Baba, email: hdobaba@kumamoto-u.ac.jp Keywords: LINE-1, methylation, esophageal cancer, prognosis, UHRF1 Received: March 24, 2016 Accepted: July 19, 2016 Published: August 05, 2016 ABSTRACT Background: Global DNA hypomethylation contributes to oncogenesis through various mechanisms. The level of long interspersed nucleotide element-1 (LINE- 1) methylation is considered a surrogate marker of global DNA methylation, and is attracting interest as a good predictor of cancer prognosis. However, the mechanism how LINE-1 (global DNA) methylation is controlled in cancer cells remains to be fully elucidated. Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) plays a crucial role in DNA methylation. UHRF1 is overexpressed in many cancers, and UHRF1 overexpression may be a mechanism underlying DNA hypomethylation in cancer cells. Nonetheless, the relationship between UHRF1, LINE-1 methylation level, and clinical outcome in esophageal squamous cell carcinoma (ESCC) remains unclear. Results: In ESCC cell lines, vector-mediated UHRF1 overexpression caused global DNA (LINE-1) hypomethylation and, conversely, UHRF1 knockdown using siRNA increased the global DNA methylation level. In ESCC tissues, UHRF1 expression was significantly associated with LINE-1 methylation levels. Furthermore, UHRF1 overexpression correlated with poor prognosis in our cohort of 160 ESCC patients. Materials and Methods: The relationships between UHRF1 expression and LINE-1 methylation level (i.e., global DNA methylation level) were investigated using ESCC tissues and cell lines. In addition, we examined the correlation between UHRF1 expression, LINE-1 methylation, and clinical outcome in patients with ESCC. Conclusions: Our results suggest that UHRF1 is a key epigenetic regulator of DNA methylation and might be a potential target for cancer treatment.

APOBEC3B belongs to the cytidine deaminase apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3) family of enzymes and induces C to T transitions of target DNA by cytidine deamination. Recently, several mutations in various cancers have been linked to APOBEC3B, suggesting a crucial role for this protein in carcinogenesis and cancer development. However, the significance of APOBEC3B in esophageal squamous cell carcinoma (ESCC) remains uncertain. In addition, the APOBEC3B immunoreactivity in cancer tissues is uncertain. Recently, we have demonstrated that PIK3CA mutation and the methylation level of long interspersed nucleotide element 1 (LINE-1) (a surrogate marker of global DNA methylation level) are prognostic markers and have crucial role on malignancy in ESCC patients. This study aims to clarify the impact of APOBEC3B on the clinical, pathological, and molecular features of ESCC. We evaluated APOBEC3B expression in ESCC and investigated the relationships among the immunoreactivity of APOBEC3B, clinical and pathological features, and the molecular features of ESCC (PIK3CA mutation, p53 expression, and LINE-1 methylation level). The immunoreactivity and mRNA level of APOBEC3B were significantly higher in cancer tissues than in noncancerous esophageal mucosae (P = 0.050). APOBEC3B expression was significantly correlated with PIK3CA mutation (P = 0.013), particularly with C to T transitions of PIK3CA (P = 0.041). Moreover, a high expression of APOBEC3B was significantly associated with LINE-1 hypomethylation (P = 0.027). Given the crucial roles of PIK3CA mutation and LINE-1 methylation levels, our findings might provide new insights into the biological mechanisms of ESCC tumorigenesis and progression.

Background There have been reported that nutritional status and tumor-infiltrating lymphocytes (TILs) are prognostic factor for esophageal cancer. Prognostic Nutritional Index (PNI) is one of the most widely used indicators for nutritional status and also shows systemic immune competence. Because TILs is related to peritumoral immune system, there may be relation between PNI and TILs. Methods Using a database of 300 curatively resected esophageal cancer from April 2005 to Jun 2013, we evaluated the relationship between PNI and TILs. PNI was calculated using serum albumin and total lymphocyte count. TILs were histologically estimated using postoperative samples. Studying the expression of CD8 and Foxp3 by immunohistochemical staining, we tried to reveal which subsets of lymphocyte were relevant to PNI. Results PNI high group (N = 198) experienced better overall survival (P Conclusion PNI was predictive prognostic marker for esophageal cancer and had relation to TILs status. It means systemic immune competence maybe affects peritumoral immune system. Among subsets of lymphocyte, CD8 positive lymphocyte had relation to PNI and was prognostic factor. In this study, Foxp3 didn’t have any relation to PNI and prognosis. Disclosure All authors have declared no conflicts of interest.

[Background] Esophageal cancer is the sixth most common cause of death from cancer worldwide. Although the development of multidisciplinary treatment for esophageal cancer, the prognosis of those patients remains poor. Recently, immunotherapy is expected as innovative therapy for several types of cancer. Indoleamine 2, 3- Dioxygenase (IDO1) is an enzyme responsible for the oxidation of tryptophan into kynurenine and implicated in immune modulation through its ability to limit T cell function and engage mechanisms of immune tolerance. IDO1 is focused as one of the immune subversion strategy and the therapy of targeting IDO1 is in clinical development for many types of cancer. IDO1 activation induces the downregulation of activating natural killer (NK) cell receptors on NK cells and inhibition of cytotoxic T cells by promotion of cell cycle arrest and apoptosis. However, the significance of IDO1 expression in esophageal cancer remains still unknown. The aim of this study is to examine the relationship between IDO1 expression and tumoral immune status, and its prognostic impact in esophageal cancer. [Methods] IDO1 expression, cluster of differentiation (CD8) expression as a marker of cytotoxic T cells, forkhead Box P3 (FOXP3) as a maker of regulatory T cells were evaluated by immunostaining in 305 clinical samples of curative resected esophageal cancer. [Result] In 305 clinical samples, high IDO1 high expression (n=71) was significantly associated with poor prognosis in overall survival(OS)(p=0.0034). In uni-and multivariate analysis, IDO1 high expression was an independent prognostic factor for OS. In those patients, CD8 low expression was also associated with poor prognosis in OS (p = 0.025). Furthermore, double immunostaning with IDO1 and CD8 revealed that CD8 expression levels was inverse correlated with IDO1 expression levels in those patients. In combination analysis of IDO1 and CD8 expression, IDO1 high expression and CD8 low expression group experienced worse overall survival (p=0.0024). Conversely, IDO1 low expression and CD8 high expression group was shown the best overall survival. In addition, IDO1 high expression group is correlate with the high counts of FOXP3 positive cells (p = 0.02). [Conclusion] IDO1 expression was associated with an unfavorable clinical outcomes by induced immune tolerance and the classification of IDO1 and CD8 expression levels might serve as a predictive biomarker in esophageal cancer. The correlation between IDO1 and FOXP3 expression suggested that IDO1 involved in immune tolerance in esophageal cancer. These findings indicate that IDO1 would be a regulator of, and candidate therapeutic target in esophageal cancer. Citation Format: Yuki Kiyozumi. IDO1 high expression is associated with a poor prognosis in patients with esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5700.

Introduction: It has become clear that macrophages are important cells forming cancer microenvironment and tumor-associated macrophages (TAM) promote their proliferation in various cancer types. In particularly, M2-type macrophages have been reported to promote the production of anti-inflammatory factors and the infiltration of regulatory T cells and suppress anti-tumor immunity, and new antitumor therapies that control tumor growth by controlling TAM have been proposed. In addition, "anti-PD-1 antibody" has attracted attention also in esophageal cancer, but the relationship between TAM and PD-L1 expression has not been clarified. Methods: A retrospective study was performed on 301 patients who underwent esophagectomy for esophageal cancer between March 2005 and June 2013. We performed immunohistochemistry and evaluation of macrophage markers (CD163 and CD204) and PD-L1. In addition, we performed in vitro experiments to clarify the cellular malignancy and relationship with PD-L1 expression. Results: Out of 301 cases of esophageal cancer, 156 cases showed CD163 high expression (51.8%) while 146 cases showed CD204 high expression (48.5%). We divided into three groups of TAM status based on CD204 and CD163 expression (CD163 and CD204 both positive: TAM status high (n=100), either one positive: moderate (n=102), both negative: low (n=99)). In TAM high group, there were more advanced cancers and adenocarcinoma cases (P Conclusion: This study suggested that TAM correlated with poor prognosis in patients with esophageal cancer. In addition, TAM correlated with invasive capacity and migratory capacity. Importantly, TAM correlated PD-L1 expression in esophageal cancer cell lines. Citation Format: Taisuke Yagi, Yoshifumi Baba, Yuki Koga, Tomoyuki Uchihara, Yuki Kiyozumi, Hiroshi Sawayama, Yukiharu Hiyoshi, Masaaki Iwatsuki, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Role of tumor-associated macrophages in esophageal cancer: PD-L1 expression and prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4742.

Background: Tumor microenvironment consists of various types of cells. Although cancer associated fibroblasts (CAFs) have been reported to enhance tumor progression and anticancer drug resistance through secretion of soluble factors so far, the crucial mechanism is not fully understood. On the other hand, advanced gastric cancer (GC) is still poor prognosis due to metastasis and recurrence despite the progression of diagnosis and treatment. The aim of this study is to identify the molecular mechanism underlying anticancer drug resistance mediated by CAFs in GC. Methods: We have isolated CAFs from surgically resected GC tissues and collected conditioned medium (CM) of CAFs. Human GC cell lines with or without CAF-CM were examined the drug sensitivity and analyzed expression of specific genes using quantitative reverse transcription PCR. We isolated extracellular vesicles (EVs) from CAF-CM by ExoQuick-TC ® and ultracentrifugal separation. We observed GC cells with confocal microscopy after the solution of stained EVs treated. We performed EVs from CAF-CM and GC-CM to mass analysis. Results: GC cells with CAF-CM showed drug resistance on normal coated plates. Moreover, we found that GC cells with CAF-CM on extracellular matrix (ECM)-coated plates showed remarkable morphological change and much higher drug resistance than those without CAF-CM. In contrast, GC cells with GC cell line-CM on ECM- coated plates did not show morphological change. We also found that EVs isolated from CAF-CM caused morphological change and drug resistance of GC cells. Moreover, we identified five proteins included in CAF-EVs as possible candidates involving in GC drug resistance by mass analysis. Gene expression analysis revealed that SNAI2, which is one of the Epithelial Mesenchymal Transition (EMT)-related gene, was significantly up-regulated in GC cells cultured with CAF-CM or EVs from CAF-CM on ECM-coated plates. Conclusion and future perspective: These findings in current study suggest that EVs from CAFs cause characteristic gene expression change and enhance drug resistance along with morphological change of GC cells. We are trying to identify the critical factor in CAF-EVs for drug resistance of GC cells. Citation Format: Tomoyuki Uchihara, Keisuke Miyake, Eri Oda, Yuki Koga, Taisuke Yagi, Daisuke Kuroda, Tsugio Eto, Yuki Kiyozumi, Kota Arima, Hiroshi Sawayama, Yukiharu Hiyoshi, Masaaki Iwatsuki, Yoshifumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba, Takatsugu Ishimoto. To identify the critical factors in extracellular vesicles derived from cancer-associated fibroblasts for drug resistance of gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5086.

Objective: Gastrointestinal stromal tumor (GIST) is a rare mesenchymal tumor originated from the interstitial cells of Cajal in the gastrointestinal tract. A significant breakthrough was achieved with the identification of the expression of the c-kit (CD117) antigen in GISTs. However, alternate oncogenic pathways have not sufficiently been understood. FBXW7 is a ubiquitin ligase that mediates ubiquitylation of oncoproteins, which is known as tumor-suppressor gene in various malignancies. Previous reports showed that FBXW7 is associated with tumor progression by regulating some oncoprotein such as c-myc and c-jun. The aim of this study is to elucidate the clinicopathological significance of FBXW7 in GIST. Methodology: (i) We examined the FBXW7 expression in GIST cells by immunostaining and correlation clinicopathological characteristics. (ii) We examined cell proliferation by Cytotoxicity assays in GIST-T1 cells suppressed by FBXW7-specific siRNA. Results: (i) In 90 cases of GIST samples with surgical resection, high FBXW7 and observed are 47 cases and low expression are 43 cases. There are significantly more large size, located small intestine, high mitotic index in low FBXW7 cases (P=0.05, P=0.01, P=0.04). There are significantly more high risk cases in modified-Fletcher classification in low FBXW7 expression cases (P=0.0008). There are significantly higher value of SUV max in high risk cases of modified-Fletcher classification and low FBXW7 expression cases (P=0.007, P=0.02). In GIST cases with distant metastasis, there are significantly more low FBXW7 expression cases than GIST cases without distant metastasis (P=0.009). FBXW7 expression is significantly inversely correlated with C-MYC expression (P=0.0004). (ii) The GIST cells suppressed FBXW7 expression by siRNA have higher proliferation rate compared to control GIST cells (P=0.02). Conclusion: We demonstrated that FBXW7 expression is correlated with modified-Fletcher classification, SUV max value, c-myc expression and cell proliferation in GIST. These results are suggesting that FBXW7 may be associated with tumor progression. These findings suggest that it is possible that FBXW7 can be a promising biomarker for disease progression of GIST. Citation Format: Yuki Koga, Masaaki Iwatsuki, Kouhei Yamashita, Yuki Kiyozumi, Hiroshi Sawayama, Yukiharu Hiyoshi, Yoshihfumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. The clinicopathoogical significance of FBXW7 expression in GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 557A.

Purpose: Gastric cancer (GC) is the fourth most commonly diagnosed cancer and the third leading cause of cancer mortality worldwide. The range of therapeutic strategies available for the treatment of GC has improved in recent decades. However, the prognosis of patients with advanced GC remains poor even after curative resection, mainly because of recurrence, such as peritoneal dissemination and liver metastases. Previous studies have shown that the interaction between cancer cells and their surrounding stroma plays an important role in tumor development. Cancer-associated fibroblasts (CAFs) have been reported to be involved in invasion and metastasis in cancers, including gastric cancer (GC), via their stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying the tumor-promoting effects and the potential of developing therapeutic targets using CXCL12/CXCR4 signaling activated by CAFs are not fully understood. Experimental Design: We first analyzed CXCL12 expression in resected GC tissues of 110 patients by immunohistochemistry (IHC). We next established primary normal fibroblasts (NFs) and CAFs from GC tissues and examined the functional differences between these primary fibroblasts using co-culture assays with GC cell lines. Furthermore, we evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF-573,228) on the invasive ability of GC cells. Results: We determined that high CXCL12 expression was significantly associated with larger tumor size, deeper tumor depth, lymphatic invasion and poor prognosis in GC. We established new primary CAFs and their adjacent NFs from resected stomach tissue in GC patients. We evaluated the change in mobility during the direct co-culture of GC cells and fibroblasts using real-time imaging. The motility of GC cells significantly increased upon direct co-culture with CAFs more than it did in cells with NFs compared with control RPMI during observation for 24 hours. The motility of GC cells with CAFs was effectively suppressed by AMD 3100 compared to GC cells with NFs. CXCL12/CXCR4 activation by CAFs mediated the clustering of integrin β1 on the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling compared with PF-573,228. Conclusions: These results suggest that CXCL12 derived from CAFs promotes invasive ability by enhancing the clustering of integrin β1 in GC cells and results in GC progression. The current data provide evidence that CXCL12/CXCR4 inhibition by AMD3100 is more efficient in suppressing GC cell invasion by not only inhibiting intracellular signaling but also by attenuating the interaction between cancer cells and the extracellular matrix compared with PF-573,228. Our present study represents the rationale for establishing a therapeutic strategy targeting CXCL12/CXCR4 signaling. Citation Format: Daisuke Izumi, Takatsugu Ishimoto, Hidetaka Sugihara, Eto Kojiro, Hiroshi Sawayama, Keisuke Miyake, Yuki Kiyozumi, Keisuke Kosumi, Ryuma Tokunaga, Kazuto Harada, Junji Kurashige, Masaaki Iwatsuki, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasive ability in gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 423. doi:10.1158/1538-7445.AM2015-423

Objectives: DNA methylation change is common epigenetic change in cancer. The promoter CpG island hypermethylation is one of the most common mechanisms by which tumor suppressor miRNAs are inactivated during tumorigenesis. However, microRNA (miRNA) regulations by epigenetic alteration, especially CpG island hypermethylation, have been not yet substantially understood in esophageal squamous cell carcinoma (ESCC). Therefore, we performed the screening of miRNAs which should be up-regulated after 5-aza-20-deoxycytidine (5-Aza) treatment in ESCC cell lines, then from up-regulated miRNAs, we focused on a suppressor miR-145-5p. Since miRNA-145-5p attracts an increasing attention as a tumor suppressor miRNA in ESCC, we evaluated miRNA-145 promoter methylation level and miRNA-145-5p expression level in ESCC Methods: The screening of miRNAs was analyzed using the Human miFinder 384HC miScript miRNA PCR Array. DNA methylation level was evaluated by using bisulfite-pyrosequencing assay. miRNA expression level was evaluated by using TaqMan quantitative real-time reverse transcription-polymerase chain reaction. miRNA-145-5p expression and its promoter methylation levels were quantified in 15 ESCC and matched 15 normal adjacent esophageal mucosa frozen tissues and in 105 formalin-fixed paraffin-embedded ESCC tissues between September 2009 and June 2012 at Kumamoto University Hospital (Kumamoto, Japan). Results Twenty miRNAs including miR-145-5p were up-regulated more than 3 fold in both TE-1 and KYSE30 after 5-Aza treatment by using miRNA PCR array analysis. After 5-Aza treatment, miRNA-145 promoter methylation level decreased and the expression level of miRNA-145-5p was significantly enhanced in 8 of 10 ESCC cell lines. The miRNA-145-5p expression levels in the cancer tissues (mean ± SD; 6.37 ± 1.47) was significantly lower than in matched normal adjacent esophageal epithelial mucosa (235.6 ± 61.9) (p = 0.0024). Moreover, the miRNA-145 promoter in the cancer tissues (41.4 ± 3.05) was significantly hyper methylated than in matched normal adjacent epithelial mucosa (mean; 57.1 ± 3.36) (p = 0.0042 by the paired t test). In 105 ESCC samples, the “high miRNA-145-5p expression level group” and “low miRNA-145-5p expression level group” experienced similar overall survival (Kaplan-Meier analysis; log rank p = 0.58) and disease-free survival (Kaplan-Meier analysis; log rank p = 0.80) Conclusions: Our results suggest that hypermethylation of the miRNA-145 promoter region might regulate miRNA-145 expression in ESCC. This is the first report on the epigenetic regulation of miRNA-145 in ESCC. However, further work is needed to assess our results and to make them more definite. Citation Format: kazuto harada, Yoshifumi Baba, Keisuke Kosumi, Ryuma Tokunaga, Daisuke Izumi, Mayuko Ouchi, Kennichi Nakamura, Yuki Kiyozumi, Junji Kurashige, Yukiharu Hiyoshi, Shiro Iwagami, Yuji Miyamoato, Yasuo Sakamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. Suppressor microRNA-145 is epigenetically regulated by promoter hypermethylation in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3132. doi:10.1158/1538-7445.AM2015-3132

Background: The incidence of esophageal adenocarcinoma (EAC) or esophagogastric junction adenocarcinoma (EGJA) has been dramatically increasing. KRAS oncogenic alterations activate multiple pathways in various types of cancer. A recent comprehensive genetic study has reported that around 20% of EAC harbor KRAS copy number gain (Dulak AM et al, 2012, Nat Genet). However, the prognostic role KRAS amplification has not been well examined in EAC/EGJA. Methods: DNA was extracted from formalin-fixed paraffin-embedded tissue of 145 EGJA (Siewert types I-III). KRAS copy number was detected by real-time quantitative PCR, and KRAS copy number gain was defined as KRAS/RNase P (as a reference) ratio ≥ 2.5. The impact of KRAS amplification was analyzed in relation to clinicopathological factors, and patient outcomes [disease free survival (DFS), and overall survival (OS)]. Multivariate cox proportional hazards model was performed adjusting clinicopathological factors. Results: KRAS amplification was observed in 40 (33.1%) cases. KRAS amplification was significantly associated with the lymph node metastasis (P = 0.0145), and was significantly associated with poorer patient outcome (P = 0.0355 in DFS, and P = 0.0067 in OS). In multivariate analysis, KRAS amplification was an independent prognostic factor for OS [multivatiate hazard ratio (HR) = 2.53; 95% confidence interval (CI), 1.22-5.25, P = 0.014] and DFS [multivatiate hazard ratio (HR) = 1.94; 95% confidence interval (CI), 1.04-3.56, P = 0.0382]. Conclusions: KRAS copy number gain may be a useful prognostic marker in EGJA. Citation Format: Kenichi Nakamura, Yu Imamura, Ryuma Tokunaga, Mayuko Ohuchi, Yuki Kiyozumi, Keisuke Kosumi, Daisuke Izumi, Kazuto Harada, Junji Kurashige, Ryuichi Karashima, Yukiharu Hiyoshi, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Hiroshi Saeki, Eiji Oki, Yoshihiko Maehara, Hideo Baba. Prognostic impact of KRAS copy number amplification in esophagogastric junction adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3861. doi:10.1158/1538-7445.AM2015-3861

Backgrounds: APOBEC3B is one of the cytidine deaminase apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3) family, and APOBEC3B can induce transitions into target DNA through cytidine deamination. To date, APOBEC attracts attention due to its functioning as part of the innate immune system by protecting against retrovirus and retrotransposon propagation. And, recent studies revealed that APOBEC3B might contribute to several mutations in various types of cancers, including cervical, breast, head and neck, lung, bladder cancers, and APOBEC3B may have a crucial role in carcinogenesis and cancer development. Esophageal squamous cell carcinoma (ESCC) reportedly has several molecular features, and we recently reported that long interspersed nucleotide element 1 (LINE-1) hypomethylation might contribute to poor prognosis and PIK3CA mutation to good prognosis in ESCC. However, there is no report which investigates the relationship between APOBEC3B expression, clinical and pathological features in ESCC. Methods: We compared the expression of APOBEC3B mRNA between cancer tissues and noncancerous esophageal mucosae by using GEO profiles and ESCC frozen tissues. In addition, we investigated the relationships between the APOBEC3B immunohistochemical expression levels, clinical and pathological features, and molecular features including p53 expression, global DNA methylation level and PIK3CA mutation. We quantified LINE-1 retrotransposons, of which a substantial portion of the human genome consists, to assess global DNA methylation. Results: APOBEC3B expression at mRNA level was significantly higher in cancer tissues than in noncancerous esophageal mucosae (P = 0.050). In the immunohistochemical study, the APOBEC3B expression of ESCC was stronger than that of noncancerous esophageal mucosa. We found no significant correlation of APOBEC3B expression with tumor size, T stage, N stage and pStage (P = 0.74, P = 0.32, P = 0.19, P = 0.23, respectively). There were no correlation between APOBEC3B expression and prognosis. There was no correlation between APOBEC3B expression and p53 expression (P = 0.23), but, interestingly, a high expression of APOBEC3B was significantly associated with LINE-1 hypomethyaltion and PIK3CA mutation (P = 0.029, P = 0.020). Conclusions: APOBEC3B high expression had significant relationships with LINE-1 hypomethylation and PIK3CA mutation, supporting that APOBEC3B might contribute to malignant behavior through the molecular features in ESCC. Citation Format: Keisuke Kosumi, Yoshifumi Baba, Takatsugu Ishimoto, Kenichi Nakamura, Kazuto Harada, Mayuko Ohuchi, Yuki Kiyozumi, Daisuke Izumi, Ryuma Tokunaga, Junji Kurashige, Ryuichi Karashima, Yukiharu Hiyoshi, Shiro Iwagami, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. The clinical significance of APOBEC3B in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4829. doi:10.1158/1538-7445.AM2015-4829

Purpose: Colorectal cancer (CRC) is the second most commonly diagnosed cancer and the third leading cause of cancer mortality in Japan. Almost 15% and 10% of patients with CRC are diagnosed with synchronous and metachronous liver metastasis, respectively. In the last decade, the therapeutic strategies including chemotherapy and molecular targeted therapy have improved. However, the prognosis of patients with CRC remains poor because of their acquirement of chemoresistance. Previous studies have shown that enhancer of zeste homologue 2 (EZH2) is associated with proliferation of various cancers. EZH2 is the catalytic subunit of Polycomb repressive complex2 (PRC2) which catalyzes the methylation of lysine 27 on histone H3 (H3K27), leading to repress the transcription of tumor-suppressor genes. In addition, high EZH2 expression is associated with the resistance to cisplatin in non-small cell lung carcinoma. However, the association between the resistance to oxaliplatin and the expression of EZH2 is unrevealed. The aim of this study is to investigate the correlation between the expression of EZH2 and response to oxaliplatin-based chemotherapy as well as survival of patients with colorectal liver metastasis (CRLM). Experimental Design: Sixty-one patients who underwent first-line oxaliplatin-based chemotherapy before resection of CRLM were enrolled in this study. Immunohistochemistry of EZH2 was performed, and then the correlation between response to chemotherapy and survival was evaluated. Results: Immunohistochemistry revealed 38 patients (62.3%) with high EZH2 expression, and 23 patients (37.7%) with low EZH2 expression. Of the 38 patients with EZH2 high expression, 15 patients (39.5%) responded to oxaliplatin-based chemotherapy with either complete response or partial response. Of the 23 patients with low EZH2 expression, 14 patients (60.9%) exhibited a response to chemotherapy. There was significant difference in response to oxaliplatin-based chemotherapy between high and low EZH2 expression patients (p = 0.04). Univariate survival analysis indicated that patients with high EZH2 expression had a lower overall survival than those with low EZH2 expression. Moreover, multivariate Cox regression analysis revealed that high EZH2 expression was an independent prognostic factor for overall survival. Kaplan-Meier survival curves also confirmed that high EZH2 expression correlates with poor prognosis in patients who underwent hepatectomy for CRLM. Conclusions: Our results indicated that prognosis of patients with high EZH2 expression who received preoperative oxaliplatin-based chemotherapy and underwent hepatectomy for CRLM was worse than those with low EZH2 expression. EZH2 may be a predictive factor for oxaliplatin-based chemotherapy response and overall survival in patients with CRLM. Citation Format: Mayuko Ohuchi, Yasuo Sakamoto, Ryuma Tokunaga, Kenichi Nakamura, Yuki Kiyozumi, Daisuke Izumi, Keisuke Kosumi, Kazuto Harada, Junji Kurashige, Yukiharu Hiyoshi, Shiro Iwagami, Yoshifumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Prognostic impact of enhancer of zeste homologue 2 (EZH2) in patients underwent hepatectomy for colorectal liver metastases who received preoperative oxaliplatin-based chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2277. doi:10.1158/1538-7445.AM2015-2277

Background Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations that activate the PI3K/AKT signaling pathway have been observed in several types of carcinoma and have been associated with patient prognosis. However, the significance of PIK3CA mutations in gastric cancer remains unclear. This retrospective study investigated the relationship between PIK3CA mutations and clinical outcomes in patients with gastric cancer. Additionally, we reviewed the rate of PIK3CA mutations in gastric cancer and the association between PIK3CA mutations and prognosis in human cancers. Methods The study included 208 patients with gastric cancer who underwent surgical resection at Kumamoto University Hospital, Japan, between January 2001 and August 2010. Mutations in PIK3CA exons 9 and 20 were quantified by pyrosequencing assays. Results PIK3CA mutations were detected in 25 (12 %) of the 208 patients. Ten patients had c.1634A > G (p.E545G), 10 had c.1624G > A (p.E542K), 13 had c.1633G > A (p.E545K), nine had c.3139C > T (p.H1047R), and 1 had c.3140A > G (p.H1047Y) mutations. PIK3CA mutations were not significantly associated with any clinical, epidemiologic, or pathologic characteristic. Kaplan–Meier analysis showed no significant differences in disease-free survival (log rank P = 0.84) and overall survival (log rank P = 0.74) between patients with and without PIK3CA mutations. Conclusions Mutations in PIK3CA did not correlate with prognosis in patients with gastric cancer, providing additional evidence for the lack of relationship between the two.