Purpose: The carcinoembryonic antigen glypican-3 (GPC3) is an ideal target of anticancer immunotherapy against hepatocellular carcinoma (HCC). In this nonrandomized, open-label, phase I clinical trial, we analyzed the safety and efficacy of GPC3 peptide vaccination in patients with advanced HCC.Experimental Design: Thirty-three patients with advanced HCC underwent GPC3 peptide vaccination (intradermal injections on days 1, 15, and 29 with dose escalation). The primary endpoint was the safety of GPC3 peptide vaccination. The secondary endpoints were immune response, as measured by IFN-g ELISPOT assay, and the clinical outcomes tumor response, time to tumor progression, and overall survival (OS).Results: GPC3 vaccination was well-tolerated. One patient showed a partial response, and 19 patients showed stable disease 2 months after initiation of treatment. Four of the 19 patients with stable disease had tumor necrosis or regression that did not meet the criteria for a partial response. Levels of the tumor markers a-fetoprotein and/or des-g-carboxy prothrombin temporarily decreased in nine patients. The GPC3 peptide vaccine induced a GPC3-specific CTL response in 30 patients. Furthermore, GPC3-specific CTL frequency after vaccination correlated with OS. OS was significantly longer in patients with high GPC3-specific CTL frequencies (N ¼ 15) than in those with low frequencies (N ¼ 18; P ¼ 0.033).Conclusions: GPC3-derived peptide vaccination was well-tolerated, and measurable immune responses and antitumor efficacy were noted. This is the first study to show that peptide-specific CTL frequency can be a predictive marker of OS in patients with HCC receiving peptide vaccination.

189 Background: Despite improvements of postoperative adjuvant therapy for resected pancreatic ductal adenocarcinoma (PDAC), its prognosis remains poor. A randomized controlled trial has begun to compare neoadjuvant chemotherapy using gemcitabine and S1 (NAC-GS) with upfront surgery (Up-S) for patients with PDAC planned resection. Methods: Patients were enrolled after the diagnosis of resectable PDAC with histological confirmation. They were randomly assigned as either NAC-GS or Up-S. In NAC-GS, gemcitabine was provided at a dose of 1 g/m2 on day 1 and 8 and oral S-1 was administered at a dose of 40 mg/m2 twice daily on 1-14 days. Patients received 2 cycles of this regimen. S-1 adjuvant for 6 months was administered for the patients with curative resection and fully recovered within 10 weeks after surgery in both arms. The primary endpoint for the phase III part was overall survival (OS); secondary endpoints included adverse events, resection rate, recurrence-free survival, residual tumor status, nodal metastases, and tumor marker kinetics. The target sample size required 163 patients (α-error 0.05; power 0.8) in each arm. The trial was conducted by the Health Labor Sciences Research Grant (H22-009) of Japan and registered with the UMIN Clinical Trials Registry as UMIN000009634. Results: From January 2013 to January 2016, 364 patients were enrolled in 57 centers (182 to NAC-GS and 182 to Up-S). Of these, two were excluded because of ineligibility, therefore 182 patients in NAC-GS and 180 in Up-S constituted the ITT analysis-set. The median OS was 36.7 months in NAC-GS and 26.6 months in Up-S; HR 0.72 (95% confidential interval 0.55-0.94; p=0.015 [stratified log-rank test]). Grade 3 or 4 adverse events frequently (72.8%) observed in NAC-GS were leukopenia or neutropenia. However, the resection rate, R0 resection rate, and morbidity of the operation were equivalent in the two groups. There was no perioperative mortality in either group. Conclusions: This phase III study demonstrated the significant survival benefits of NAC-GS treatment. Therefore, the results indicated that neoadjuvant chemotherapy could be a new standard for patients with resectable PDAC. Clinical trial information: UMIN000009634.

The relationship between overexpression of glypican (GPC)-3 that is specific for hepatocellular carcinoma (HCC) and the prognosis has not yet been clarified. We attempted to determine the expression profile of GPC3 in association with the clinicopathological factors by immunohistochemical analysis in HCC patients and investigated the potential prognostic value of GPC3 by comparing the survival rate between the GPC3-positive and GPC3-negative HCC patients. Primary HCC tissue samples (n = 107) obtained from patients who had undergone hepatectomy between 2000 and 2001 were analyzed. GPC3 expression was less frequently observed in welldifferentiated HCC than in moderately and poorly differentiated HCC, the difference in the frequency being statistically significant. GPC3-positive HCC patients had a significantly lower 5-year survival rate than the GPC3-negative HCC patients (54.5 vs 87.7%, P = 0.031). Among 80 of the 107 (74.6%) patients with initial treatment who underwent hepatectomy, none of GPC3-negative HCC patients (n = 16, 20.0%) died during the follow-up period. No deaths were noted in the GPC3-negative HCC patients among the 71 (88.7%) patients with moderately and poorly differentiated HCC. Multivariate analysis identified GPC3 expression (P = 0.034) as an independent prognostic factor for the overall survival. We showed that GPC3 expression is correlated with a poor prognosis in HCC patients. (Cancer Sci 2009; 100: 1403-1407) H epatocellular carcinoma (HCC) is one of the most common malignancies and is ranked as the third most common cause of cancer-related death worldwide. HCC is generally associated with a poor prognosis, the 5-year survival rate after surgery has been reported to be as low as 25-39%, and systemic therapy with cytotoxic agents provides only marginal benefit.(1) Even in those patients in whom the tumor has been successfully removed, the 2-year recurrence rate can be as high as 50%. (2,3) Several clinicopathological factors including poor levels of differentiation of the cancer cells, large size of the tumor, portal venous invasion, and intrahepatic metastasis have been shown to contribute to the poor prognosis in patients of HCC. Despite the critical need for better methods for the diagnosis and treatment of HCC, the mechanisms underlying the development of HCC remain unclear.Glypican (GPC)-3 was discovered as a potential serological and histochemical marker that is specific for HCC. GPC3 is a member of the glypican family and belongs to a group of heparan sulfate proteoglycans bound to the outer surface of the cell membrane through a glycosylphosphatidylinositol anchor. (4) In mammals, this family comprises six members, GPC1 to GPC6. GPC are released from the cell surface by a lipase called Notum to regulate the signaling of Wnts, Hedgehogs, fibroblast growth factors, and bone morphogenetic proteins.(5-9) Depending on the context, their functions exerted may either be stimulatory or inhibitory through these pathways. GPC3 has been detected in the placenta and fetal liver, but not in othe...

Background-Remnant lipoproteins are atherogenic, but assays of remnants have not been available in routine clinical laboratories because of the lack of practical and validated methods. A simple and reliable method for such an assay, using an immunochemical approach, has recently been developed. This study prospectively examined whether remnant lipoprotein levels in fasting serum, measured by our method, may have prognostic value in patients with coronary artery disease (CAD). Methods and Results-Remnant lipoprotein levels in fasting serum were measured in 135 patients with CAD by an immunoaffinity mixed gel containing anti-apolipoprotein (apo) A-1 and anti-apoB-100 monoclonal antibodies. Patients were followed up for Յ36 months until occurrence of 1 of the following clinical coronary events: recurrent or refractory angina pectoris requiring coronary revascularization, nonfatal myocardial infarction, or cardiac death. Kaplan-Meier analysis demonstrated a significantly higher probability of developing coronary events in patients with the highest tertile of remnant levels (Ͼ5.1 mg cholesterol/dL; 75th percentile of distribution of remnant levels) than in those with the lowest tertile of remnant levels (Յ3.3 mg cholesterol/dL; 50th percentile of the distribution). Higher levels of remnants were a significant and independent predictor of developing coronary events in multivariate Cox hazard analysis including the following covariates: extent of coronary artery stenosis, age, sex, smoking, hypertension, diabetes mellitus, hypercholesterolemia, low HDL cholesterol, and hypertriglyceridemia. Conclusions-Higher levels of remnant lipoproteins in fasting serum predict future coronary events in patients with CAD independently of other risk factors. Thus, measurement of fasting remnant levels, assessed by the current immunoseparation method, may be helpful in assessment of CAD risk. (Circulation. 1999;99:2858-2860.)

Remnant lipoprotein levels were independently associated with abnormal endothelium-dependent vasomotor function in large and resistance coronary arteries in humans, indicating that remnant lipoproteins may impair endothelial vasomotor function in human coronary arteries. The decrease in coronary nitric oxide bioactivity may be responsible in part for the inhibitory effects of remnant lipoproteins.

Background-The circulating levels of secretory nonpancreatic type II phospholipase A 2 (sPLA 2 ) are increased in various chronic inflammatory diseases and the increase in the levels correlates with the disease severity. sPLA 2 may possibly play a role in atherogenesis and is highly expressed in atherosclerotic arterial walls that are known to have inflammatory features. Thus, this study prospectively examined whether circulating levels of sPLA 2 may have a significant risk and prognostic values in patients with coronary artery disease (CAD). Methods and Results-Plasma levels of sPLA 2 were measured in 142 patients with CAD and in 93 control subjects by a radioimmunoassay. The sPLA 2 levels had a significant and positive relations with serum levels of C-reactive protein, a marker of systemic inflammation, and with the number of the traditional coronary risk factors associated with individuals. Multivariate logistic regression analysis showed that higher levels of sPLA 2 (Ͼ246 ng/dL; 75th percentile of sPLA 2 distribution in controls) were a significant and independent risk factor for the presence of CAD. In multivariate Cox hazard analysis, the higher levels of sPLA 2 were a significant predictor of developing coronary events (ie, coronary revascularization, myocardial infarction, coronary death) during a 2-year follow-up period in patients with CAD independent of other risk factors, including CRP levels, an established inflammatory predictor. Conclusions-The increase in circulating levels of sPLA 2 is a significant risk factor for the presence of CAD and predicts clinical coronary events independent of other risk factors in patients with CAD; these results may reflect possible relation of sPLA 2 levels with inflammatory activity in atherosclerotic arteries. (Circulation. 1999;100:1280-1284.)

Background-This study was designed to evaluate the plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in patients with unstable angina and investigate whether there is a relationship between these levels and unfavorable outcome. Methods and Results-The plasma TF and free TFPI antigen levels were determined in plasma samples taken from 51 patients with unstable angina, 56 with stable exertional angina, and 55 with chest pain syndrome. The plasma TF and free TFPI antigen levels were higher in the unstable angina group than in the stable exertional angina and chest pain syndrome group. There was a good correlation between TF and TFPI. We established borderline as maximum level in the patients with chest pain syndrome. Seven patients (of the 22 in the high TF group) required revascularization to control their unstable angina during in-hospital stay. On the other hand, only 1 of the 29 patients in the low TF group required myocardial revascularization. Four patients of the 14 patients in the high free TFPI group required myocardial revascularization during in-hospital stay, and 4 of the 37 patients in the low free TFPI group required myocardial revascularization. We compared the TF and free TFPI levels between the cardiac event (ϩ) group and cardiac event (Ϫ) group. TF levels were significantly higher in the cardiac event (ϩ) group than in the cardiac event (Ϫ) group. Conclusions-We have demonstrated that not only the plasma TF levels but also the plasma-free TFPI levels are elevated in patients with unstable angina. Patients with unstable angina and heightened TF and free TFPI are at increased risk for unfavorable outcomes. The heightened TF level was a more important predictor in patients with unstable angina.

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