A phase I study of combination vaccine treatment of five therapeutic epitope-peptides for metastatic colorectal cancer; safety, immunological response, and clinical outcome.

BACKGROUND: To evaluate the safety of combination vaccine treatment of multiple peptides, phase I clinical trial was conducted for patients with advanced colorectal cancer using five novel HLA-A*2402-restricted peptides, three peptides derived from oncoantigens, ring finger protein 43 (RNF43), 34 kDa-translocase of the outer mitochondrial membrane (TOMM34), and insulin-like growth factor-II mRNA binding protein 3 (KOC1), and the remaining two from angiogenesis factors, vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2. METHODS: Eighteen HLA- A*2402-positive colorectal cancer patients who had failed to standard therapy were enrolled in this study. 0.5 mg, 1.0 mg or 3.0 mg each of the peptides was mixed with incomplete Freund's adjuvant and then subcutaneously injected at five separated sites once a week. We also examined possible effect of a single site injection of "the cocktail of 5 peptides" on the immunological responses. ELISPOT assay was performed before and after vaccinations in the schedule of every 4 weeks. RESULTS: The vaccine treatment using multiple peptides was well tolerated without any severe treatment-associated systemic adverse events. Dose-dependent induction of peptide-specific cytotoxic T lymphocytes was observed. The single injection of "peptides cocktail" did not diminish the immunological responses. Regarding the clinical outcome, one patient achieved complete response and 6 patients revealed stable disease for 4 to 7 months. The median overall survival time (MST) was 13.5 months. Patients, in which we detected induction of cytotoxic T lymphocytes specific to 3 or more peptides, revealed significantly better prognosis (MST; 27.8 months) than those with poorer immune responses (MST; 3.7 months) (p = 0.032). CONCLUSION: Our cancer vaccine treatment using multiple peptides is a promising approach for advanced colorectal cancer with the minimum risk of systemic adverse reactions. CLINICAL TRIAL REGISTRATION: UMIN-CTR number UMIN000004948.

Photon pair generation in a silicon micro-ring resonator with reverse bias enhancement.

Photon sources are fundamental components for any quantum photonic technology. The ability to generate high count-rate and low-noise correlated photon pairs via spontaneous parametric down-conversion using bulk crystals has been the cornerstone of modern quantum optics. However, future practical quantum technologies will require a scalable integration approach, and waveguide-based photon sources with high-count rate and low-noise characteristics will be an essential part of chip-based quantum technologies. Here, we demonstrate photon pair generation through spontaneous four-wave mixing in a silicon micro-ring resonator, reporting separately a maximum coincidence-to-accidental (CAR) ratio of 602 ± 37 (for a generation rate of 827kHz), and a maximum photon pair generation rate of 123 MHz ± 11 kHz (with a CAR value of 37). To overcome free-carrier related performance degradations we have investigated reverse biased p-i-n structures, demonstrating an improvement in the pair generation rate by a factor of up to 2 with negligible impact on CAR.

Objective evaluation of tongue diagnosis ability using a tongue diagnosis e-learning/e-assessment system based on a standardized tongue image database.

Background: In Kampo medicine, tongue examination is used to diagnose the pathological condition "Sho," but an objective evaluation method for its diagnostic ability has not been established. We constructed a tongue diagnosis electronic learning and evaluation system based on a standardized tongue image database. Purpose: This study aims to verify the practicality of this assessment system by evaluating the tongue diagnosis ability of Kampo specialists (KSs), medical professionals, and students. Methods: In the first study, we analyzed the answer data of 15 KSs in an 80-question tongue diagnosis test that assesses eight aspects of tongue findings and evaluated the (i) test score, (ii) test difficulty and discrimination index, (iii) diagnostic consistency, and (iv) diagnostic match rate between KSs. In the second study, we administered a 20-question common Kampo test and analyzed the answer data of 107 medical professionals and 56 students that assessed the tongue color discrimination ability and evaluated the (v) correct answer rate, (vi) test difficulty, and (vii) factors related to the correct answer rate. Result: In the first study, the average test score was 62.2 ± 10.7 points. Twenty-eight questions were difficult (correct answer rate, <50%), 34 were moderate (50%-85%), and 18 were easy (≥85%). Regarding intrarater reliability, the average diagnostic match rate of five KSs involved in database construction was 0.66 ± 0.08, and as for interrater reliability, the diagnostic match rate between the 15 KSs was 0.52 (95% confidence interval, 0.38-0.65) for Gwet's agreement coefficient 1, and the degree of the match rate was moderate. In the second study, the difficulty level of questions was moderate, with a correct rate of 81.3% for medical professionals and 82.1% for students. The discrimination index was good for medical professionals (0.35) and poor for students (0.06). Among medical professionals, the correct answer group of this question had a significantly higher total score on the Kampo common test than the incorrect answer group (85.3 ± 8.4 points vs. 75.8 ± 11.8 points, p < 0.01). Conclusion: This system can objectively evaluate tongue diagnosis ability and has high practicality. Utilizing this system can be expected to contribute to improving learners' tongue diagnosis ability and standardization of tongue diagnosis.

Inter-rater reliability of self-reported response on foreskin status in questionnaire among Japanese adult men.

OBJECTIVES: To determine whether foreskin status is a measurable marker for evaluating the effect of the foreskin on sexually transmitted infections. METHODS: Inter-rater comparison of the responses on foreskin status and circumcision in a self-report questionnaire with the findings of a physical examination by an experienced well-trained urologist was performed for patients who visited a healthcare facility in Kanagawa, Japan. Foreskin status was defined using a five-point graphical scale based on the degree to which the foreskin covers the foreskin and the glans penis in a non-erectile condition: type 1, a fully exposed glans penis; types 2-4, the glans penis partly covered by the foreskin and type 5, phimosis. Linear weighted κ and per cent agreement were used to evaluate the reliability of responses. RESULTS: Among 188 participants who were evaluated about their foreskin status, linear weighted κ and per cent agreement were 0.74% and 68.4%, respectively. Linear weighted κ improved from 0.74 to 0.80 when the number of categories was changed to three. All the self-reported responses on circumcision were in agreement with the findings of the physical examination. Seventeen participants (9.0%) had been circumcised, and among them, three (17.6%) had approximately one-half of their glans penis covered by the foreskin. In 90 among the 171 uncircumcised participants (52.6%), the foreskin did not cover the glans penis. CONCLUSIONS: The self-reported response on foreskin status in this questionnaire has sufficient reliability to replace physical examination, and this questionnaire can facilitate further studies about the effect of foreskin on sexually transmitted infections.

Construction of a Standardized Tongue Image Database for Diagnostic Education: Development of a Tongue Diagnosis e-Learning System.

Tongue examination is an important diagnostic method for judging pathological conditions in Kampo (traditional Japanese medicine), but it is not easy for beginners to learn the diagnostic technique. One reason is that there are few objective diagnostic criteria for tongue examination findings, and the educational method for tongue examination is not standardized in Japan, warranting the need for a tongue image database for e-learning systems that could dramatically improve the efficiency of education. Therefore, we constructed a database comprising tongue images whose findings were determined on the basis of votes given by five Kampo medicine specialists (KMSs) and confirmed the educational usefulness of the database for tongue diagnosis e-learning systems. The study was conducted in the following five steps: development of a tongue imaging collection system, collection of tongue images, evaluation and annotation of tongue images, development of a tongue diagnosis e-learning system, and verification of the educational usefulness of this system. Five KMSs evaluated the tongue images obtained from 125 participants in the following eight aspects: (i) tongue body size, (ii) tongue body color, (iii) tongue body dryness and wetness, (iv) tooth marks on the edge of the tongue, (v) cracks on the surface of the tongue, (vi) thickness of tongue coating, (vii) color of tongue coating, and (viii) dryness and wetness of tongue coating. Medical students (MSs) were given a tongue diagnosis test using an e-learning system after a lecture on tongue diagnosis. The cumulative and individual match rates (%) (individual match rates of 100% (5/5), 80% (4/5), and 60% (3/5) are shown in parentheses, respectively) were as follows: (i) tongue body size: 92.8 (26.4/26.4/40.0); (ii) tongue body color: 83.2 (10.4/20.8/52.0); (iii) tongue body dryness and wetness: 88.8 (13.6/34.4/40.8); (iv) tooth marks on the edge of the tongue: 88.8 (6.4/35.2/47.2); (v) cracks on the surface of the tongue: 96.8 (24.0/35.2/37.6); (vi) thickness of tongue coating: 84.8 (7.2/21.6/56.0); (vii) color of tongue coating: 88.0 (15.2/37.6/35.2); and (viii) dryness and wetness of tongue coating: 74.4 (4.8/19.2/50.4). The test showed that the tongue diagnosis ability of MSs who attended a lecture on tongue diagnosis was almost the same as that of KMSs. We successfully constructed a tongue image database standardized for training specialists on tongue diagnosis and confirmed the educational usefulness of the e-learning system using a database. This database will contribute to the standardization and popularization of Kampo education.

On-chip optical interconnection by using integrated III-V laser diode and photodetector with silicon waveguide.

On-chip integration of III-V laser diodes and photodetectors with silicon nanowire waveguides is demonstrated. Through flip-chip bonding of GaInNAs/GaAs laser diodes directly onto the silicon substrate, efficient heat dissipation was realized and characteristic temperatures as high as 132K were achieved. Spot-size converters for the laser-to-waveguide coupling were used, with efficiencies greater than 60%. The photodetectors were fabricated by bonding of InGaAs/InP wafers directly to silicon waveguides and formation of metal-semiconductor-metal structures, giving responsivities as high as 0.74 A/W. Both laser diode and the photodetector were integrated with a single silicon waveguide to demonstrate a complete on-chip optical transmission link.

Methylation of multiple genes as molecular markers for diagnosis of a small, well-differentiated hepatocellular carcinoma.

The current study was conducted to identify robust methylation markers and their combinations that may prove useful for the diagnosis of early hepatocellular carcinoma (HCC). To achieve this, we performed in silico CpG mapping, direct sequencing and pyrosequencing after bisulfite treatment, and quantitative methylation-specific PCR (MSP) in HCC and non-HCC liver tissues. In the filtering group (25 HCCs), our direct sequencing analysis showed that, among the 12 methylation genes listed by in silico CpG mapping, 7 genes (RASSF1A, CCND2, SPINT2, RUNX3, GSTP1, APC and CFTR) were aberrantly methylated in stages I and II HCCs. In the validation group (20 pairs of HCCs and the corresponding non-tumor liver tissues), pyrosequencing analysis confirmed that the 7 genes were aberrantly and strongly methylated in early HCCs, but not in any of the corresponding non- tumor liver tissues (p < 0.00001). The results obtained using our novel quantitative MSP assay correlated well with those observed using the pyrosequencing analysis. Notably, in MSP assay, RASSF1A showed the most robust performance for the discrimination of HCC and non-HCC liver tissues. Furthermore, a combination of RASSF1A, CCND2 and SPINT2 showed 89-95% sensitivity, 91-100% specificity and 89-97% accuracy in discriminating between HCC and non-HCC tissues, and correctly diagnosed all early HCCs. These results indicate that the combination of these 3 genes may aid in the accurate diagnosis of early HCC.

Integration of GaN/AlN all-optical switch with SiN/AlN waveguide utilizing spot-size conversion.

Spot-size converters for an all-optical switch utilizing the intersubband transition in GaN/AlN multiple quantum wells are studied with the purpose of reducing operation power by improving the coupling efficiency between the input fiber and the switch. With a stair-like spot-size converter, the absorption saturation of 5 dB is achieved with a pulse energy of 25 pJ. The switch is integrated with a SiN/AlN waveguide and spot-size converters, and the structure provides the possibility of an integration of the switch with other functional devices. To further improve the coupling loss between the waveguide and the switch, triangular-shaped converters are investigated, demonstrating losses as low as 2 dB/facet.

Decreased ID2 promotes metastatic potentials of hepatocellular carcinoma by altering secretion of vascular endothelial growth factor.

PURPOSE: We aimed to explore the molecular and biological functions of Inhibitor of DNA binding/differentiation 2 (ID2), which was found to be responsible for portal vein invasion of hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: We measured ID2 mRNA levels in 92 HCC patients by real-time reverse transcription-PCR and examined the relation to clinicopathologic features. To clarify the precise roles of ID2, we did in vitro analysis with expression vectors and small interfering RNAs. Effects of ID2 on cell invasive potential and expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha were analyzed by Matrigel-coated invasion chamber, ELISA, and Western blot analysis, respectively. RESULTS: ID2 mRNA level correlated inversely with portal vein invasion (P < 0.001), tumor-node-metastasis stage (P < 0.001), tumor size (P < 0.001), and early intrahepatic recurrence (P < 0.05). When limited to a cohort of hepatitis C virus-related HCCs, patients with low levels of ID2 had significantly shorter disease-free survival time than those with high levels of ID2. Invasive potential of cells transfected with ID2 expression vector was lower than that of empty vector-transfected cells. Cells overexpressing ID2 also showed decreased VEGF secretion and hypoxia-inducible factor-1alpha protein levels. The results of ID2-knockdown experiments were opposite to those of ID2 overexpression experiments. CONCLUSIONS: On the basis of our clinical and in vitro data, we suggest that ID2 plays a significant role in the metastatic process during progression of HCC. This action might be explained, at least in part, by altered cell mobility due to decreased secretion of VEGF.

High serum levels of vascular endothelial growth factor after hepatectomy are associated with poor prognosis in hepatocellular carcinoma.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) reportedly affects the metastatic potential of tumors. We investigated in patients whether association exits between perioperative serum VEGF levels and recurrence of hepatocellular carcinoma (HCC). METHODOLOGY: Thirty-two patients who underwent curative resection for HCC were enrolled in this study. Blood samples were obtained at 6 points during the perioperative period: preoperative day and, postoperative days 1, 2, 3, 7 and 14). Serum VEGF levels were measured by enzyme-linked immunosorbent assay. We divided the 32 hepatocellular carcinoma patients into a high VEGF group (preoperative serum level > or = 100 pg/ ml, n = 10) and a low VEGF group (preoperative serum level < 100 pg/ml, n = 22). RESULTS: During a median follow-up period of 27.1 months, the cancer recurred in 20 of the 32 patients. Disease-free survival in the high VEGF group was significantly poorer than that in the low VEGF group (p < 0.05). Serum VEGF levels in the high VEGF group remained significantly higher than those in the low VEGF group after hepatectomy (p < 0.05). Serum VEGF levels in the recurrent group were also significantly higher than those in the non recurrent group at preoperative day, postoperative days 2, 3 and 7 (p < 0.05). CONCLUSIONS: High serum VEGF levels during perioperative period may be a risk factor for intrahepatic recurrence after complete resection for HCC.

Translational microarray systems for outcome prediction of hepatocellular carcinoma.

DNA microarray technology has revolutionized our understanding of the molecular basis of hepatocellular carcinoma (HCC), one of the most fatal human cancers with a high recurrence rate. Many researchers have used DNA microarray technology to reclassify HCC with respect to metastatic potential and to develop predictors for the outcome of HCC. However, developed predictors have reached the level only of small retrospective studies, and their current status is far from that required for clinical use. This is due to the lack of transparent data, the high cost and data instability associated with the high dimensionality of the technique, the infancy of bioinformatics, and the complicated nature of recurrent HCC. This comprehensive review summarizes: (i) class comparison studies to identify genes or pathways involved in HCC metastasis (ii) class discovery studies that have resulted in the identification of a new molecular subclass of HCC with respect to metastasis, and (iii) class prediction studies to develop multidimensional predictors for HCC outcome. We also discuss issues that need to be addressed so that the power of array-based predictors can be estimated prospectively in large independent cohorts of HCC patients.

Identification of novel aberrant methylation of BASP1 and SRD5A2 for early diagnosis of hepatocellular carcinoma by genome-wide search.

A genome-wide study using expression profiles of 12,600 genes was conducted to identify methylated genes that could be used for early diagnosis of hepatocellular carcinoma (HCC). Of the 12,600 genes examined, we identified 23 genes with significantly lower expression levels in HCC tissues than in non-HCC liver tissues by our statistical and CpG mapping tests. Of these 23 genes, methylation analysis by direct sequencing with bisulfite treatment determined 4 genes that were aberrantly methylated in 20 HCC samples of TNM stages I and II. Further methylation analysis of the 4 genes by quantitative sequencing with 20 HCCs and the corresponding non-tumor liver tissues from an independent cohort of HCC patients revealed that 2 genes, BASP1 and SRD5A2, were aberrantly methylated in only HCC tissues, though not in any corresponding non-tumor liver tissues. Notably, in the cohort we found that BASP1 or SRD5A2 were aberrantly methylated when a cut-off value of 30% in the methylation rate was used, in all cases of 11 HCCs of TNM stages I and II, of 10 well-differentiated HCCs and of 4 small HCCs <2 cm in maximum diameter, but in none of the 20 corresponding non-HCC livers. Methylation-specific PCR for BASP1 and SRD5A2 reproduced the same results observed by direct sequencing. These results indicate that BASP1 and SRD5A2 might serve as useful biomarkers for early diagnosis of HCC.

Glycolysis module activated by hypoxia-inducible factor 1alpha is related to the aggressive phenotype of hepatocellular carcinoma.

An increased level of glycolysis, an intracellular hallmark of neoplasms, enables cancer cells to survive under various conditions. To elucidate the role of increased glycolysis in the progression of hepatocellular carcinoma (HCC), we investigated the associations between the expression patterns of 14 glycolysis-related genes and clinicopathologic factors in 60 HCCs by using pooled transcriptome data. We then evaluated the therapeutic efficacy of the knockdown of ENO1, which is encoded by a glycolysis-related gene, in HCC cells. Among the 14 genes, levels of 8 genes (GPI, ALDOA, TPI1, GAPD, PGK, PGAM, ENO1 and PKM), all of which can be transcriptionally activated by hypoxia-inducible factor 1alpha (HIF-1alpha), were significantly higher in HCC with venous invasion (VI) than in HCC without VI. Our cluster analysis showed that HCC patients with activation of the 8 HIF-1alpha-regulated genes had significantly shorter overall survival (P=0.023) than did HCC patients without increased expression levels of these genes. The association between the levels of ENO1 and VI was confirmed in an independent sample set of 49 HCCs by real-time reverse-transcription PCR. The knockdown of ENO1 by small-interfering RNA significantly inhibited the proliferation of an HCC cell line (HLE cells) in both the glucose-rich and glucose-free conditions, accompanied by a decreased S phase and increased G2/M phase of the cell cycle. Collectively, these data suggest that activation of an HIF-1alpha-regulated glycolysis module is closely related to the aggressive phenotype of HCC, and that ENO1, a glycolysis module gene, might serve as a new target to circumvent HCC metastasis.

A three-gene predictor for early intrahepatic recurrence of hepatocellular carcinoma after curative hepatectomy.

We previously developed a DNA microarray-based system that out-performs traditionally used clinical parameters for prediction of early intrahepatic recurrence (IHR) of hepatocellular carcinoma (HCC). Because DNA microarray is too expensive for daily clinical use, we used a quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) to develop a lower-cost predictor for early IHR. From the 12 early IHR-related genes integrated in the previous predictor, we selected 6 genes whose levels showed the strongest association between data from the 2 distinct DNA microarray platforms with the same sample set. Expression of these 6 genes relative to that of GAPDH was measured by QRT-PCR in 82 HCCs. Of the 82 HCCs, 39 and 43 were assigned to training and independent test sets, respectively. By searching all combinations (n=2-6) of the 6 genes, we found an optimal combination of 3 genes (HLADRA, DDX17 and LAPTM5) that minimized the leave-one-out error for prediction of early IHR in the training set. The 3-gene predictor constructed with the Fisher linear classifier correctly predicted early IHR or non-recurrence in 35 (81.4%) of 43 HCCs in the independent test set and had a high positive predictive value of 72.7% and a high negative predictive value of 84.4%. Multivariate analysis with the stepwise logistic regression showed that the 3-gene predictor [F(x)<0] was an independent risk factor for early IHR (risk ratio, 13.6; p=0.006), indicating its potential as an easy-to-use predictor for accurate prediction of early IHR of HCC.

Relation between serum levels of cell-free DNA and inflammation status in hepatitis C virus-related hepatocellular carcinoma.

Our study revealed that the level of circulating cell-free DNA (cfDNA) is increased in the serum of patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). To gain insight into the mechanism underlying this phenomenon, we examined the association between cfDNA levels and various clinicopathological factors in 96 patients with HCV-related HCC and 99 non-HCC patients with HCV. Using pooled DNA microarray data, we profiled the expression patterns of inflammatory cytokine genes in 14 primary tumors from the group of HCC patients. We found that there were positive associations between the cfDNA level, aspartate aminotransferase levels and the number of leukocytes and neutrophils in patients with HCV-related HCC but not in non-HCC patients with HCV. The serum cfDNA level was not associated with other clinicopathological factors in HCC or non-HCC patients. A cluster analysis based on the inflammatory cytokine gene data revealed that HCCs with a high serum cfDNA level had increased levels of several inflammatory cytokine genes, suggesting that the serum cfDNA level is associated with the inflammatory status in primary tumors in HCV-related HCC.

A new prognostic model for hepatocellular carcinoma recurrence after curative hepatectomy.

We previously reported the effectiveness of the product of tumor number and size (NxS factor) for the prognosis of hepatocellular carcinoma (HCC) in patients following hepatectomy. The present study aimed to propose a new score based on the NxS factor to predict HCC recurrence following hepatectomy. A total of 406 patients who underwent hepatectomy for HCC at Osaka University Graduate School of Medicine were retrospectively analyzed to develop the new score. Among clinicopathological factors, including the NxS factor, the marker subset that achieved the best performance for prediction of early recurrence was assessed, and a prognostic model for HCC recurrence after curative hepatectomy (REACH) was developed. As the validation set, 425 patients who underwent hepatectomy for HCC at Yamaguchi University Graduate School of Medicine and Shimonoseki Medical Center were analyzed, and the prognostic ability of the REACH score was compared with that of well-known staging systems. Following analysis, the REACH score was constructed using six covariates (NxS factor, microscopic hepatic vein invasion, differentiation, serum albumin, platelet count and indocyanine green retention rate at 15 min). In the validation set, the REACH score predicted early recurrence in 73 of 81 samples, with a sensitivity of 89% and a specificity of 58%. The area under the curve (AUC) of the receiver operating characteristic curve of the REACH score was 0.78 and 0.74, respectively, for 1- and 2-year recurrence after hepatectomy; each AUC was higher than that of any of the other staging systems. Survival analysis indicated the REACH score had the best predictive value in disease-free and overall survival. The present findings demonstrated that the REACH score may be used to classify patients with HCC into high- and low-risk of recurrence, and to predict subsequent survival following hepatic resection.

Expression levels of UL16 binding protein 1 and natural killer group 2 member D affect overall survival in patients with gastric cancer following gastrectomy.

UL16 binding protein 1 (ULBP1) expressed on the tumor cell surface binds to the natural killer group 2 member D (NKG2D) receptor presenting on natural killer (NK), cluster of differentiation (CD)8+ T, and γ δ T cells. However, the roles of ULBP1 and NKG2D expression and associated immune responses in gastric cancer are unclear. The present study investigated the associations between ULBP1 and NKG2D expression and clinical outcomes in patients with gastric cancer. The levels of ULBP1 and NKG2D expression were examined in human gastric cancer cell lines and gastric cancer tissues from 98 patients who underwent surgery from 2004 to 2008. MKN-74 cells expressed ULBP1 with ULBP2, -5, or -6. NKG2D was expressed at a higher level following activation of T cells and NK cells. Among the tissue sections positive for NKG2D expression, 6 patients were positive for CD8 and CD56. In all tissues, NKG2D-expressing cells were typically aCD8+ T cells. Patients with NKG2D expression in tumors exhibited significantly longer overall survival (OS) compared with patients without NKG2D expression in tumors (P=0.0217). The longest OS was observed in patients positive for ULBP1 and NKG2D, whereas the shortest OS was observed in patients negative for ULBP1 and NKG2D. The interaction between ULBP1 and NKG2D may improve OS in patients with gastric cancer, and may have applications in immunotherapy for the induction of adaptive immunity in patients with cancer. Additionally, ULBP1 and NKG2D may be useful as prognostic biomarkers in gastric cancer.

Resampling based on geographic patterns of hepatitis virus infection reveals a common gene signature for early intrahepatic recurrence of hepatocellular carcinoma.

The majority of hepatocellular carcinomas (HCCs) correlated with infection by either hepatitis B or C virus (HBV or HCV) showing various geographic distributions, making it impossible to identify common gene signatures responsible for HCC recurrence. In this study we performed in silico resampling analysis of DNA microarray data that can reproduce virtually the geographic distribution pattern of HBV and HCV in 6 representative geographic regions. With the use of the Fisher ratio, genes associated with early intrahepatic recurrence of HCC within 1 year of surgery were identified in the 6 geographic virtual cohorts, each consisting of 1,000 virtual samples. The top 100 genes among each virtual cohort were compared. Many human leukocyte antigen (HLA) family genes were common among the 6 geographic virtual cohorts, suggesting that this gene family represents the pathway most responsible for early intrahepatic recurrence of HCC worldwide. This resampling approach is useful for identifying common pathways involved in various aspects of HCC.

Protein level of apolipoprotein E increased in human hepatocellular carcinoma.

Many reports suggest that hepatic steatosis leads to hepatocellular carcinoma (HCC), including hepatitis C virus or non-alcoholic steatohepatitis. Proteomic study of tumor tissues from HCC patients, focusing on apolipoprotein (apo) of apoA1, apoB100 and apoE, was performed by immunoblotting. Although the significant changes of apoA1 or apoB100 could not be shown statistically, the immunoblotting showed the increase in protein level of apoE in the tumor tissues of 88% of patients without increase of apoE gene expression and serum level. These results suggest the accumulation of apoE by impaired secretion. Moreover, immunoblot analysis on two-dimensional electrophoresis showed a strong possibility that sialylated forms of apoE also were increased in tumorous tissues of HCC. ApoE level in tumorous tissues is frequently elevated and may be a good histological marker for HCC.

Identification of ID2 associated with invasion of hepatitis C virus-related hepatocellular carcinoma by gene expression profile.

Portal vein invasion (PVI) is a hallmark of metastatic potential of hepatocellular carcinoma (HCC) and is frequently found at a stage of moderately differentiated HCC. To identify genes involved in PVI of HCC associated with hepatitis C virus (HCV), we performed a comprehensive analysis of 12,600 genes in 35 moderately differentiated HCV-related HCCs by DNA microarray. Our supervised learning method identified 35 genes involved in PVI. Among the 35 identified genes, we focused on the inhibitor of DNA binding 2 (ID2), because it encodes a liver-rich dominant-negative helix-loop-helix protein. The microarray results for ID2 were reproduced by quantitative real-time reverse transcription (QRT)-PCR and Western blot analyses. In an independent set of HCV-related HCCs (n=28) and HCV-unrelated HCCs (n=14), our QRT-PCR showed that decrease in ID2 mRNA levels were associated with PVI in HCV-related HCC but not HCV-unrelated HCC. In conclusion, our results strongly suggest that ID2 plays an important role in PVI process of HCV-related HCC.