Antibody-based proteomics to identify an apoptosis signature for early recurrence of hepatocellular carcinoma.

BACKGROUND: Early recurrence after surgical resection is a hallmark of poor prognosis in hepatocellular carcinoma (HCC). To determine the proteomic background of early recurrence of HCC, we focused on apoptosis-related proteins. METHODS: Surgically resected tumor tissues were obtained from 80 patients, including HCC tumor tissues, non-tumor tissues, and normal liver tissues. These samples were grouped in the discovery and validation sample sets. The expression level of 192 apoptosis-related proteins was monitored using 247 commercially available antibodies and western blotting. The intensity of protein bands was compared between the tumor and non-tumor tissues as well as between the patients who had recurrence within 2 years after surgery and those who did not. RESULTS: In the first screening, we used pooled samples. The intensity of 53 protein bands detected by 37 unique antibodies was higher in tumor tissues compared with normal liver tissues, especially tumor tissues from patients who had recurrence within 2 years after surgery. In the second screening, we examined individual samples used to make the pooled samples. Among the selected bands and antibodies, the intensity of 18 protein bands detected by 11 antibodies was higher in tumor tissues compared with that in normal tissues, especially tumor tissues from the patients with early recurrence after surgery. For the third screening, we examined the samples from newly enrolled patients using these 11 antibodies. Eighteen protein bands detected by six antibodies were selected by using the same criteria. The corresponding antigens included ERK1, PKG, Apaf1, BclX, phosphorylated c-abl, and PIASx1/2. CONCLUSIONS: We screened 192 apoptosis-related proteins using specific antibodies and western blotting. We identified 6 apoptosis-related proteins associated with carcinogenesis and early recurrence in HCC. The biological and clinical significance of the identified proteins are worth further investigation.

Global PROTOMAP profiling to search for biomarkers of early-recurrent hepatocellular carcinoma.

This study used global protein expression profiling to search for biomarkers to predict early recurrent hepatocellular carcinoma (HCC). HCC tissues surgically resected from patients with or without recurrence within 2 years (early recurrent) after surgery were compared with adjacent nontumor tissue and with normal liver tissue. We used the PROTOMAP strategy for comparative profiling, which integrates denaturing polyacrylamide gel electrophoresis migratory rates and high-resolution, semiquantitative mass-spectrometry-based identification of in-gel-digested tryptic peptides. PROTOMAP allows examination of global changes in the size, topography, and abundance of proteins in complex tissue samples. This approach identified 8438 unique proteins from 45 708 nonredundant peptides and generated a proteome-wide map of changes in expression and proteolytic events potentially induced by intrinsic apoptotic/necrotic pathways. In the early recurrent HCC tissue, 87 proteins were differentially expressed (≥20-fold) relative to the other tissues, 46 of which were up-regulated or specifically proteolyzed and 41 of which were down-regulated. This data set consisted of proteins that fell into various functional categories, including signal transduction and cell organization and, notably, the major catalytic pathways responsible for liver function, such as the urea cycle and detoxification metabolism. We found that aberrant proteolysis appeared to occur frequently during recurrence of HCC in several key signal transducers, including STAT1 and δ-catenin. Further investigation of these proteins will facilitate the development of novel clinical applications.

Macrophage-capping protein as a tissue biomarker for prediction of response to gemcitabine treatment and prognosis in cholangiocarcinoma.

Cholangiocarcinoma is one of the deadliest malignancies worldwide. Recent studies reported that treatment with gemcitabine was effective in prolonging survival. However, as the treatment only benefited a limited subset of patients, selection of patients before treatment is required. To discover biomarkers predictive of the response to gemcitabine treatment in cholangiocarcinoma, we examined the proteome of three types of material resource; ten cell lines, nine xenografts and nine surgically resected primary tumors from patients who exhibited different response to gemcitabine treatment. Two-dimensional difference gel electrophoresis generated quantitative protein expression profiles including 3571 protein spots. We detected 172 protein spots with significant correlation with response to gemcitabine treatment. All proteins corresponding to these 172 protein spots were identified by mass spectrometry. We found that the macrophage-capping protein (CapG) was associated with response to gemcitabin treatment in all three types of material source. Immunohistochemical validation in an additional set of 196 cholangiocarcinoma cases revealed that CapG expression was associated with lymphatic invasion status and overall survival. Multivariate analysis showed that CapG protein expression was an independent prognostic factor for overall survival. In conclusion, CapG was identified as a novel candidate biomarker to predict response to gemcitabine treatment and survival in cholangiocarcinoma.

Establishment of six new human biliary tract carcinoma cell lines and identification of MAGEH1 as a candidate biomarker for predicting the efficacy of gemcitabine treatment.

The aim of this study was to establish new biliary tract carcinoma (BTC) cell lines and identify predictive biomarkers for the potential effectiveness of gemcitabine therapy. Surgical specimens of BTC were transplanted directly into immunodeficient mice to establish xenografts, then subjected to in vitro cell culture. The gemcitabine sensitivity of each cell line was determined and compared with the genome-wide gene expression profile. A new predictive biomarker candidate was validated using an additional cohort of gemcitabine-treated BTC cases. From 55 BTC cases, we established 19 xenografts and six new cell lines. Based on their gemcitabine sensitivity, 10 BTC cell lines (including six new and four publicly available ones) were clearly categorized into two groups, and MAGEH1 mRNA expression in the tumor cells showed a significant negative correlation with their sensitivity to gemcitabine. Immunohistochemically, MAGEH1 protein was detected in three (50%) out of six sensitive cell lines, and four (100%) out of four resistant cell lines. In the validation cohort of gemcitabine-treated recurrence cases, patients were categorized into "effective" and "non-effective" groups according to the RECIST guidelines for assessment of chemotherapeutic effects. MAGEH1 protein expression was detected in two (40%) out of five "effective" cases and all four (100%) "non-effective" cases. We have established a new BTC bioresource that covers a wide range of biological features, including drug sensitivity, and is linked with clinical information. Negative expression of MAGEH1 protein serves as a potential predictive marker for the effectiveness of gemcitabine therapy in BTC.

An analysis of risk factors for pancreatic fistula after pancreaticoduodenectomy: clinical impact of bile juice infection on day 1.

BACKGROUND: Postoperative pancreatic fistula (POPF) is a most striking complication after pancreatic resection. The objective of this study is to reveal the risk factors for POPF defined by the international study group after pancreaticoduodenectomy in a Japanese high-volume center. METHODS: During the recent 4 years, 220 patients underwent pancreaticoduodenectomies. In patients of obstructive jaundice, preoperative biliary drainage was performed by percutaneous (n = 71) and/or retrograde (n = 38) approach. Pancreaticojejunostomy was performed using either duct-to-mucosa anastomosis (n = 180) or dunking method (n = 40). Risk factors for POPF (grade B or grade C POPF by international definition) were evaluated using univariate and multivariate analyses. RESULTS: POPF was found in 109 (50%) patients; grade A in 45 (21%), grade B in 54 (25%), and grade C in 10 patients (5%). One patient died of intra-abdominal hemorrhage caused by POPF. Univariate and multivariate analyses revealed that independent risk factors for grade B or grade C POPF were the size of the main pancreatic duct (<3 mm; relative risk (RR), 3.3; p = 0.002), body mass index (> or =20, RR 2.5, p = 0.03), and bile juice infection on day 1 (RR, 2.2; p = 0.04). The performance of biliary drainage or method of pancreaticojejunostomy was not a significant risk factor for POPF. Bile juice infection on day 1 was significantly associated with retrograde biliary drainage (p < 0.001). CONCLUSIONS: Bile juice infection on day 1 was a significant risk factor for grade B or grade C POPF after pancreaticoduodenectomy. Although the performance or the status of biliary drainage itself was not a risk factor for POPF, percutaneous biliary drainage might be advantageous against retrograde drainage to reduce the risk of biliary infection.

Predictive value of the time-intensity curves on dynamic contrast-enhanced magnetic resonance imaging for lymphatic spreading in breast cancer.

PURPOSE: Dynamic contrast-enhanced magnetic resonance imaging (CE-MRI) has emerged as a promising diagnostic modality in various breast cancer treatments. However, little is known about the correlation between the pattern of time to signal intensity curves (TIC) on the CE-MRI and clinicopathologic features. This study was designed to investigate these correlations and evaluate the predictive value of TIC on CE-MRI in order to identify high-risk patients. METHODS: Between 2001 and 2003, 101 lesions were evaluated to detect malignancy on CE-MRI in 101 women who were suspected of having breast tumors based on either clinical findings or conventional imaging studies. Moreover, the clinicopathologic findings were compared with the pattern of TIC for the 69 surgically treated malignant lesions. RESULTS: In detecting malignancy, the sensitivity, specificity, and accuracy were 78.7%, 88.5%, and 81.2%, respectively, in the 101 breast lesions. Especially for the 69 surgically treated malignant lesions, in comparison with breast cancer tumors with the benign pattern of TIC, the breast cancer tumors with a malignant pattern were found more frequently in lymphatic invasion (P < 0.01) and lymph node metastasis (P < 0.005), although no statistical correlation regarding the histological type, tumor size, vascular invasion, extensive intraductal component, hormone receptor status, or pathological stage was noted between the two groups. According to a logistic regression model, lymph node metastasis was found to be a significant independent variable. CONCLUSION: The pattern of TIC could be used to predict lymphatic spreading associated with lymph node metastasis prior to surgery as well as to detect malignancy. Therefore, a more detailed evaluation should be made to identify the presence of lymphatic spreading in patients with a malignant pattern of TIC.