Yuki Kitano

C5a receptor (CD88) promotes motility and invasiveness of gastric cancer by activating RhoA

Purpose Anaphylatoxin C5a is a strong chemoattractant of the complement system that binds the C5a receptor (C5aR). The expression of C5aR is associated with poor prognosis in several cancers. However, the role of C5aR in gastric cancer (GC) is unknown. The aim of this study was to examine the role of C5aR on GC cell motility and invasion. Experimental Design The mechanism of invasion via C5aR was assessed by analyzing cytoskeletal rearrangement and RhoA activity after C5a treatment. Moreover, we investigated the relationship between C5aR expression and the prognosis of GC patients. Results Two human GC cell lines (MKN1 and MKN7) had high C5aR expression. An invasion assay revealed that C5a stimulation promoted the invasive ability of MKN1 and MKN7 cells and that this was suppressed by knockdown of C5aR using siRNA or a C5aR-antagonist. Moreover, overexpression of C5aR in GC cells enhanced the conversion of RhoA-guanosine diphosphate (RhoA-GDP) to RhoA-guanosine triphosphate (RhoA-GTP) after C5a stimulation and caused morphological changes, including increased expression of stress fibers and filopodia. Examination of tumor specimens from 100 patients with GC revealed that high C5aR expression (35 of 100 samples, 35.0%) was associated with increased invasion depth, vascular invasion and advanced stage. The 5-year overall survival of patients with high or low C5aR expression was 58.2% and 88.5%, respectively (p=0.008). Conclusions This study is the first to demonstrate that C5aR promotes GC cell invasion by activating RhoA and is associated with a poor prognosis in GC patients. Therefore, this study provides a biomarker for GC patients who require an advanced therapeutic strategy.

Tumour-infiltrating inflammatory and immune cells in patients with extrahepatic cholangiocarcinoma

Background:Inflammation and immune characteristics of the tumour microenvironment have therapeutic significance. The aim of this study was to investigate the clinical impact on disease progression in human extrahepatic cholangiocarcinoma (ECC).Methods:A total of 114 consecutive ECC patients with curative resection between 2000 and 2014 were enrolled. Tumour infiltrating CD66b+ neutrophils (TANs; tumour associated neutrophils), CD163+ M2 macrophages (TAMs; tumour associated macrophages), CD8+ T cells, and FOXP3+ regulatory T cells (Tregs) were assayed by immunohistochemistry, and their relationships with patient clinicopathological characteristics and prognosis were evaluated.Results:Tumour associated neutrophils were inversely correlated with CD8+ T cells (P=0.0001) and positively correlated with Tregs (P=0.001). High TANs (P=0.01), low CD8+ T cells (P=0.02), and high Tregs (P=0.04) were significantly associated with poor overall survival (OS). A high-risk signature, derived from integration of intratumoural inflammatory and immune cells, was significantly associated with poor recurrence-free survival (P=0.01) and OS (P=0.0008). A high-risk signature was correlated with postoperative distant metastases. Furthermore, a high-risk signature was related to the resistance to gemcitabine-based chemotherapy used after recurrence.Conclusions:Our data showed that tumour infiltrating inflammatory and immune cells may play a pivotal role in ECC progression and a high-risk signature predicted poor prognosis in ECC patients.

Preoperative platelet-to-lymphocyte ratio can predict recurrence beyond the Milan criteria after hepatectomy for patients with hepatocellular carcinoma.

Postoperative recurrence beyond the Milan criteria is a poor prognostic factor for patients with hepatocellular carcinoma (HCC) treated with various therapies. We investigated the most useful inflammation‐based prognostic score for predicting recurrence beyond the Milan criteria after initial liver resection.

Nrf2 promotes oesophageal cancer cell proliferation via metabolic reprogramming and detoxification of reactive oxygen species: Nrf2 and glutathione metabolism in oesophageal cancer

Cancer cells consume a large amount of energy and maintain high levels of anabolism to promote cell proliferation via metabolic reprogramming. Nuclear factor erythroid 2‐related factor 2 (Nrf2; NFE2L2) is a master transcription regulator of stress responses and promotes metabolic reprogramming to support cell proliferation in various types of cancer. As oesophageal cancer is one of the most aggressive gastrointestinal cancers, we aimed to clarify the effect of Nrf2 on metabolic reprogramming in oesophageal cancer. The relationship between Nrf2 expression and clinical outcome was evaluated using a database comprising 201 oesophageal cancers. Using in vitro assays and metabolome analysis, we examined the mechanism by which Nrf2 affects malignant phenotype. High‐level immunohistochemical expression of Nrf2 was significantly associated with poor recurrence‐free survival (HR = 2.67, p = 0.0004) and overall survival (HR = 2.90, p < 0.0001) in oesophageal cancer patients. In an in vitro assay with siRNA in TE‐11 cells, which showed high Nrf2 expression, Nrf2 depletion significantly decreased cell growth and enhanced G1 cell cycle arrest and apoptosis. In addition, reactive oxygen species (ROS) were not removed by detoxification via the Nrf2 pathway, with concomitant induction of the p38 mitogen‐activated protein kinase pathway. The metabolome analysis showed that Nrf2 strongly promoted metabolic reprogramming to glutathione metabolism, which synthesizes the essential fuels for cancer progression. Furthermore, metabolome analysis using oesophageal cancer specimens confirmed that samples displaying high Nrf2 expression promoted glutathione synthesis. Metabolic reprogramming to glutathione metabolism, and ROS detoxification by activation of Nrf2, enhanced cancer progression and led to a poor clinical outcome in oesophageal cancer patients. Copyright

Number of acinar cells at the pancreatic stump predicts pancreatic fistula after pancreaticoduodenectomy

PurposeTo establish if the number of pancreatic acinar cells at the pancreatic cut end is a predictor of postoperative pancreatic fistula (POPF).MethodsThe number of acinar cells was assessed histologically in 121 consecutive patients who underwent pancreaticoduodenectomy (PD) between April, 2012 and July, 2016.ResultsPOPF developed in 23 of the 121 patients. Univariate analysis revealed that male sex, long operating time, high volume of blood loss, soft remnant pancreas, large pancreatic duct, and the number of pancreatic acinar cells were significantly associated with POPF. Multivariate analysis revealed that male sex (p = 0.022) and the number of pancreatic acinar cells (p < 0.0001) were independently associated with POPF. In the receiver operating characteristic (ROC) curve analysis, the area under curve was 0.83895 when the cut off value of the number of pancreatic acinar cells to predict POPF was 890. Sensitivity and specificity of the number of pancreatic acinar cells were 82.6 and 77.6%, respectively.ConclusionsA large number of pancreatic acinar cells at the cut end of the stump is predictive of POPF after PD. Although POPF is associated with multiple factors and the number of acinar cells is only one of these, our study is the first to confirm this common intuition of surgeons, which has not been assessed definitively before.

Prognostic value of LINE-1 methylation level in 321 patients with primary liver cancer including hepatocellular carcinoma and intrahepatic cholangiocarcinoma

Background The methylation level of long interspersed nucleotide element-1 (LINE-1) is a good surrogate marker of the global DNA methylation level. The relationship between LINE-1 methylation level and prognosis in primary liver cancer (PLC) patients remains unclear. Results LINE-1 methylation levels were significantly lower in HCC and cHCC-CC tissues, but not in ICC tissues, than those in noncancerous liver parenchyma (HCC: p < 0.0001; cHCC-CC: p < 0.001; and ICC: p = 0.053). HCC cases with hypomethylated LINE-1 had significantly shorter relapse-free survival (RFS) (log-rank, p = 0.008); however, this was not observed for the cHCC-CC or ICC cases. Multivariate Cox regression analysis revealed a significantly higher HCC recurrence rate in the group with hypomethylated LINE-1 (hazard ratio, 1.62; 95% confidence interval, 1.06–2.58; p = 0.025). Conclusions The genome-wide DNA hypomethylation status estimated via LINE-1 methylation levels might be indicative of poor RFS in patients with HCC but not ICC or cHCC-CC. Methods We evaluated the level of LINE-1 methylation in 321 cases of curatively resected PLC {231 hepatocellular carcinoma (HCC), 19 combined hepatocellular and cholangiocarcinoma (cHCC-CC) and 71 intrahepatic cholangiocarcinoma (ICC)} via pyrosequencing of formalin-fixed paraffin-embedded (FFPE) tissues and examined its prognostic value.

Predicting Poorly Differentiated Hepatocellular Carcinoma that Meets the Milan Criteria

Background/Aim: Poorly differentiated hepatocellular carcinoma (HCC) is a malignant phenotype following radiofrequency ablation, but not liver resection. This study aimed to identify prognostic parameters that could predict poorly differentiated HCC. Patients and Methods: Between 2007-2014, 158 HCC patients undergoing liver resection were enrolled that not the Milan criteria. Laboratory data were measured including three tumor markers and inflammatory factors (neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio, and monocyte/lymphocyte ratio. Preoperative parameters to predict poorly differentiated HCC were assessed by multivariate logistic regression analysis. Results: Poorly differentiated HCC was observed in 28 (17.7%) patients. In multivariate analysis, two or three positive tumor markers and high NLR (≥2.33) were independent predictors of poorly differentiated HCC. Recurrence-free and overall survival were comparable despite these significant predictors. Conclusion: The preoperative status of two or three positive tumor markers and high NLR facilitated selecting HCC patients with poorly differentiated disease, which will assist making therapeutic decisions for HCC patients.

Microvascular invasion of single small hepatocellular carcinoma ≤3 cm: Predictors and optimal treatments

Small hepatocellular carcinomas (HCC ≤3 cm) are generally considered to have low malignant potential; however, some of them display pathological microvascular invasion (MVI).

Hepatobiliary and Pancreatic: Hepatocellular carcinoma developed with angiomyolipoma

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver, and it is most commonly caused by a multistep hepatocarcinogenesis preceded by cirrhosis or viral hepatitis. Angiomyolipoma (AML) is considered a rare and oftenmisdiagnosed as HCC to be resected. We herein present a patient who had an extremely rare case of HCC, which newly detected in a large AML. A 42-year-old man with lung cancer was admitted to our hospital and suspected to have AML in left lobe by computed tomography (CT) and positron emission tomography/CT (PET-CT) (Fig. 1a). The patient underwent a left upper lobectomy for lung cancer, and following which, he underwent chemotherapy with cisplatin and vinorelbine. According to our policy and because the diameter of the AML tumor exceeded 5 cm, we intended to perform a hepatectomy, provided that the lung cancer did not recur. Two years after the surgery for lung cancer, a new solid nodule, with a diameter of 4 cm, was detected within the AML tumor by CT. The nodule was suspected to be HCC because serum alpha-fetoprotein (AFP) level was 10.8 ng/mL and AFP-L3 was 74.3%. CT showed the nodule as an enhanced lesion within AML (Fig. 1b). PET-CT showed it as an Fluorodeoxyglucose (FDG)-accumulated mass, with a maximum standardized uptake value of 9.7 (Fig. 1c). We diagnosed this newly developed mass as HCC; therefore, we performed a left hemi-hepatectomy, which included resection of AML. The resected specimen included a large hepatic mass measuring 15 × 10 cm, with a separate encapsulated nodule of 5.5 × 3.0 cm in size (Fig. 1d). During histologic examination, the large hepatic mass was diagnosed as AML and the inner small nodule was diagnosed as moderately differentiated HCC with trabecular arrangement (Fig. 1e). Histologically, HCC and AML elements were not intermingling. In addition, HCC nodule was completely included within the AML mass, and there is no continuity between HCC and non-tumorous liver tissue. Immunohistochemical staining results were as follows: human melanoma black 45 and alpha-smooth muscle actin were positive for AML, and glypican-3 (GP3) was positive for HCC (Fig. 1f–h). Angiomyolipoma is a mesenchymal tumor; however, there is a wide spectrum of opinions about the origin of AML. Bonetti et al. have suggested that AMLmay originate from perivascular epithelioid cells, making it part of a family of perivascular epithelioid cell tumors. Yang et al. reported that renal AML was derived from adhesive cells possessing the characteristics of mesenchymal stem cells (MSCs). Although we do not know how HCC develop within AML, we have three hypotheses. First, HCC developed from hepatocytes in AML. Second, HCC developed from hepatocytes that migrated from the normal liver to AML. Finally, HCC developed from MSCs in AML. In our case, we could not confirm the presence of hepatocytes in AML or the HCC nodule derived from normal liver, making it difficult to support our first and the second hypothesis. Because MSCs can differentiate into hepatocytes, if a hepatic AML contains MSCs, HCC can develop fromMSCs within AML. Therefore, we consider the final hypothesis to be possible. In renal AML, Inomoto et al. reported renal cell carcinoma within AML and made reference to the possibility that the renal cell carcinoma originated in the AML. In summary, we report an unusual case of HCC, which developed with a hepatic AML.

Abstract 1770: The association between sarcopenia and cellular senescence of cancer associated fibroblast in pancreatic cancer

Background: Sarcopenia is a syndrome that is characterized by progressive and generalized loss of skeletal muscle mass and strength. Several recent studies have shown that sarcopenia has prognostic significance for patients with malignant disease, including cancers of the esophagus, colon, pancreas and liver. On the other hand, cellular senescence is gaining increased attention from clinicians and researchers, yet incompletely understood role in the development of malignant disease. Previous studies reported that Caveolin-1 expression in cancer associated fibroblast is associated with cancer progression, and it is known in recent years that caveolin-1 plays a major role in controlling cellular senescence. Therefore, we hypothesized that sarcopenia causes cellular senescence of cancer associated fibroblast (CAF). The aim of this study is to clarify the between sarcopenia and cellular senescence of CAF through Caveolin-1 in pancreatic cancer. Methods: All consecutive patients with pancreatic cancer underwent curative resection between January 2004 and December 2014 were enrolled in this retrospective study. Skeletal muscle and visceral fat amount at the third lumbar vertebra (L3) in the inferior direction were quantified using enhanced computed tomography. The patients were divided into two groups, with and without sarcopenia, based on Japan Society of Hepatology guidelines for sarcopenia. Next, Caveolin-1 expression was analyzed by immunohistochemistry (IHC). Moreover, we investigated the relationship between sarcopenia and Caveolin-1 expression. Results: Forty-one (31.5%) of 130 patients were identified with sarcopenia. Kaplan-Meier analysis showed that patients with sarcopenia had a significantly worse cancer specific survival than patients without sarcopenia (3-year-survival: 37.2% vs 50.4%, respectively, p = 0.038). While, a high level of Caveolin-1 expression was detected in 29.2% (38/130) of our samples. High Caveolin-1 expression in CAF significantly correlated with poor prognoses with respect to overall survival (log-rank p = 0.014) and disease-free survival (log-rank p = 0.0015). However, there was no relationship between sarcopenia and IHC score of Caveolin-1 expression in CAF. Conclusion: The present result suggested that sarcopenia and Caveolin-1 expression of CAF in patient with pancreatic cancer is respectively associated with poor prognosis, but not associated each other. Now, we focus on Caveolin-1 expression of CAF, and establish primary cultures of CAF from the pancreatic cancer tissues and examine its function. Citation Format: Kensuke Yamamura, Yo-ichi Yamashita, Yuki Kitano, Kota Arima, Takayoshi Kaida, Tatsunori Miyata, Shigeki Nakagawa, Kosuke Mima, Katsunori Imai, Daisuke Hashimoto, Akira Chikamoto, Hideo Baba. The association between sarcopenia and cellular senescence of cancer associated fibroblast in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1770. doi:10.1158/1538-7445.AM2017-1770