Combination gemcitabine plus S-1 versus gemcitabine plus cisplatin for advanced/recurrent biliary tract cancer: the FUGA-BT (JCOG1113) randomized phase III clinical trial.

BACKGROUND: Gemcitabine plus cisplatin (GC) is the standard treatment of advanced biliary tract cancer (BTC); however, it causes nausea, vomiting, and anorexia, and requires hydration. Gemcitabine plus S-1 (GS) reportedly has equal to, or better, efficacy and an acceptable toxicity profile. We aimed to confirm the non-inferiority of GS to GC for patients with advanced/recurrent BTC in terms of overall survival (OS). PATIENTS AND METHODS: We undertook a phase III randomized trial in 33 institutions in Japan. Eligibility criteria included chemotherapy-naïve patients with recurrent or unresectable BTC, an Eastern Cooperative Oncology Group Performance Status of 0 - 1, and adequate organ function. The calculated sample size was 350 with a one-sided α of 5%, a power of 80%, and non-inferiority margin hazard ratio (HR) of 1.155. The primary end point was OS, while the secondary end points included progression-free survival (PFS), response rate (RR), adverse events (AEs), and clinically significant AEs defined as grade ≥2 fatigue, anorexia, nausea, vomiting, oral mucositis, or diarrhea. RESULTS: Between May 2013 and March 2016, 354 patients were enrolled. GS was found to be non-inferior to GC [median OS: 13.4 months with GC and 15.1 months with GS, HR, 0.945; 90% confidence interval (CI), 0.78-1.15; P = 0.046 for non-inferiority]. The median PFS was 5.8 months with GC and 6.8 months with GS (HR 0.86; 95% CI 0.70-1.07). The RR was 32.4% with GC and 29.8% with GS. Both treatments were generally well-tolerated. Clinically significant AEs were observed in 35.1% of patients in the GC arm and 29.9% in the GS arm. CONCLUSIONS: GS, which does not require hydration, should be considered a new, convenient standard of care option for patients with advanced/recurrent BTC. CLINICAL TRIAL NUMBER: This trial has been registered with the UMIN Clinical Trials Registry (http://www.umin.ac.jp/ctr/index.htm), number UMIN000010667.

A randomized phase II study of S-1 plus oral leucovorin versus S-1 monotherapy in patients with gemcitabine-refractory advanced pancreatic cancer.

BACKGROUND: We evaluated the efficacy and toxicity of adding oral leucovorin (LV) to S-1 when compared with S-1 monotherapy in patients with gemcitabine-refractory pancreatic cancer (PC). PATIENTS AND METHODS: Gemcitabine-refractory PC patients were randomly assigned in a 1:1 ratio to receive S-1 at 40, 50, or 60 mg according to body surface area plus LV 25 mg, both given orally twice daily for 1 week, repeated every 2 weeks (SL group), or S-1 monotherapy at the same dose as the SL group for 4 weeks, repeated every 6 weeks (S-1 group). The primary end point was progression-free survival (PFS). RESULTS: Among 142 patients enrolled, 140 were eligible for efficacy assessment (SL: n = 69 and S-1: n = 71). PFS was significantly longer in the SL group than in the S-1 group [median PFS, 3.8 versus 2.7 months; hazard ratio (HR), 0.56; 95% confidence interval (CI), 0.37-0.85; P = 0.003]). The disease control rate was significantly higher in the SL group than in the S-1 group (91% versus 72%; P = 0.004). Overall survival (OS) was similar in both groups (median OS, 6.3 versus 6.1 months; HR, 0.82; 95% CI, 0.54-1.22; P = 0.463). After adjusting for patient background factors in a multivariate analysis, OS tended to be better in the SL group (HR, 0.71; 95% CI, 0.47-1.07; P = 0.099). Both treatments were well tolerated, although gastrointestinal toxicities were slightly more severe in the SL group. CONCLUSION: The addition of LV to S-1 significantly improved PFS in patients with gemcitabine-refractory advanced PC, and a phase III trial has been initiated in a similar setting. CLINICAL TRIALS NUMBER: Japan Pharmaceutical Information Center: JapicCTI-111554.

Sorafenib plus hepatic arterial infusion chemotherapy with cisplatin versus sorafenib for advanced hepatocellular carcinoma: randomized phase II trial.

BACKGROUND: Sorafenib (Sor) is acknowledged as a standard therapy for advanced hepatocellular carcinoma (HCC). This trial was conducted to evaluate the effect of addition of hepatic arterial infusion chemotherapy with cisplatin (SorCDDP) to Sor for the treatment of advanced HCC. PATIENTS AND METHODS: We conducted a multicenter open-labeled randomized phase II trial in chemo-naïve patients with advanced HCC with Child-Pugh scores of 5-7. Eligible patients were randomly assigned 2:1 to receive SorCDDP (sorafenib: 400 mg bid; cisplatin: 65 mg/m2, day 1, every 4-6 weeks) or Sor (400 mg bid). The primary end point was overall survival. RESULTS: A total of 108 patients were randomized (Sor, n = 42; SorCDDP, n = 66). The median survival in the Sor and SorCDDP arms were 8.7 and 10.6 months, respectively [stratified hazard ratio (95% confidence interval), 0.60 (0.38-0.96), P = 0.031]. The median time to progression and the response rate were, respectively, 2.8 months and 7.3% in the Sor arm and 3.1 months and 21.7% in the SorCDDP arm. The adverse events were more frequent in the SorCDDP arm than in the Sor arm, but well-tolerated. CONCLUSION: SorCDDP yielded favorable overall survival when compared with Sor in patients with advanced HCC. CLINICAL TRIAL REGISTRATION: UMIN-CTR (http://www.umin.ac.jp/ctr/index-j.htm), identification number: UMIN000005703.

Randomised phase II trial of S-1 plus oxaliplatin vs S-1 in patients with gemcitabine-refractory pancreatic cancer.

BACKGROUND: This randomised, open-label, multicenter phase II study compared progression-free survival (PFS) of S-1 plus oxaliplatin (SOX) with that of S-1 alone in patients with gemcitabine-refractory pancreatic cancer. METHODS: Patients with confirmed progressive disease following the first-line treatment with a gemcitabine-based regimen were randomised to receive either S-1 (80/100/120 mg day(-1) based on body surface area (BSA), orally, days 1-28, every 6 weeks) or SOX (S-1 80/100/120 mg day(-1) based on BSA, orally, days 1-14, plus oxaliplatin 100 mg m(-2), intravenously, day 1, every 3 weeks). The primary end point was PFS. RESULTS: Between January 2009 and July 2010, 271 patients were randomly allocated to either S-1 (n=135) or SOX (n=136). Median PFS for S-1 and SOX were 2.8 and 3.0 months, respectively (hazard ratio (HR)=0.84; 95% confidence interval (CI), 0.65-1.08; stratified log-rank test P=0.18). Median overall survival (OS) was 6.9 vs 7.4 months (HR=1.03; 95% CI, 0.79-1.34; stratified log-rank test P=0.82). The response rate (RR) was 11.5% vs 20.9% (P=0.04). The major grade 3/4 toxicities (S-1 and SOX) were neutropenia (11.4% and 8.1%), thrombocytopenia (4.5% and 10.3%) and anorexia (12.9% and 14.7%). CONCLUSIONS: Although SOX showed an advantage in RR, it provided no significant improvement in PFS or OS compared with S-1 alone.

The significance of gastrectomy in advanced gastric cancer patients with non-curative factors.

BACKGROUND: The role of gastrectomy in the treatment of advanced gastric cancer patients with non-curative factors remains controversial. We investigated prognostic factors and evaluated the role of gastrectomy in such patients. PATIENTS AND METHODS: Eighty-eight advanced gastric cancer patients with non-curative factors were prospectively studied. The patients were categorized into the following two groups: Group A: 52 patients who underwent gastrectomy and subsequently received chemotherapy, Group B: 36 patients who received chemotherapy alone. RESULTS: The median survival times of group A and B patients were 351 and 182 days, respectively (p=0.008). Multivariate analysis showed that gastrectomy was the only positive independent prognostic factor, with no effect on the results of chemotherapy. There was no significant difference in the duration of hospital stay between patients of the two groups, while significantly longer maintenance of oral intake was observed for group A. CONCLUSION: In advanced gastric cancer patients with non-curative factors, gastrectomy was beneficial for survival with longer maintenance of oral intake.

Updated results from GEST study: a randomized, three-arm phase III study for advanced pancreatic cancer.

PURPOSE: The GEST study showed non-inferiority of S-1 but not superiority of gemcitabine plus S-1 (GS) to gemcitabine alone for overall survival with the data by the cut-off date of 31st July in 2010 for chemo-naïve patients with advanced pancreatic cancer. We considered it important to determine whether S-1 maintains non-inferiority after a long-term follow-up in the GEST study and to obtain a firm positive conclusion. In addition, it may be an interesting challenge to explore the efficacious profile of GS in the long-term follow-up study. Using the data from the follow-up period, background and efficacy in patients from Taiwan and Japan, as well as the rates of tumor shrinkage in locally advanced and metastatic patients (Waterfall plot) were also analyzed. METHODS: The results of the primary analysis were reconfirmed, and subset analysis of overall survival and progression-free survival was performed based on the overall survival data updated by the cut-off date of 31st July in 2011. RESULTS: The median follow-up period was 29.8 months, and 795 deaths occurred (95.6%). The median overall survival was 8.8 months for gemcitabine, 9.7 months for S-1 (hazard ratio [HR], 0.96; 97.5% confidence interval [CI], 0.79-1.17), and 9.9 months for GS (HR 0.91; 97.5% CI 0.75-1.11). In patients with performance status (PS) 0, the median overall survival was 9.8 months for gemcitabine, 10.9 months for S-1, and 10.5 months for GS. In patients with PS 1, the median overall survival was 6.2 months for gemcitabine, 6.3 months for S-1, and 9.6 months for GS. CONCLUSION: Our survey reconfirmed the non-inferiority of S-1 to gemcitabine and showed S-1 can be used as one of the standard treatment options for advanced pancreatic cancer. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00498225.

Overexpression of small GTP-binding protein RhoA promotes invasion of tumor cells.

Adhesion of tumor cells to host cell layers and subsequent migration are pivotal steps in cancer invasion and metastasis. The small GTP-binding protein RhoA controls cell adhesion and motility through organization of the actin cytoskeleton and regulation of actomyosin contractility. Cultured rat MM1 hepatoma cells migrate through a mesothelial cell monolayer in vitro in a serum-dependent, RhoA-mediated manner (K. Yoshioka et al., J. Biol. Chem., 273: 5146-5154, 1998). Furthermore, the ROCK family of RhoA-associated serine-threonine protein kinases is involved in this migration, and an inhibitor for these kinases effectively inhibits the invasion of MM1 cells in vitro and in vivo (K. Itoh et al., Nat. Med., 5: 221-225, 1999). Although there have been no reports of genetic alterations directly affecting RhoA in human cancer, the expression level of RhoA in tumors has been several times higher than that of surrounding normal tissue; RhoA was especially highly expressed in the metastatic region. To determine whether RhoA is activated by its overexpression, we made stable transfectants of MM1 cells expressing various levels of wild-type human RhoA. These transfectants showed promoted invasive ability in vitro in the absence and presence of 1-oleoyl-lysophosphatidic acid, marked adherence to the plastic culture dish with scattered shape, elevated phosphorylation of Mr 20,000 myosin light chain, and translocation of RhoA protein from the cytosol to the membrane. All of these phenotypes were similar to those of active RhoA transfectants, correlated with the expression level of RhoA and reversed by the treatment of the cells with Clostridium botulinum exoenzyme C3 ADP-ribosyltransferase. In addition, overexpression of wild-type RhoA in MM1 cells also conferred invasive ability in vivo after the cells were transplanted into the syngeneic rats. Thus, high expression of RhoA in the cell facilitates the translocation of this protein to the membrane, where it is activated, resulting in the stimulation of the RhoA-ROCK-actomyosin system, leading to invasion.

Increased expression of sialyl Lewisx antigen correlates with poor survival in patients with colorectal carcinoma: clinicopathological and immunohistochemical study.

We have previously shown that sialyl Lewisx antigen (sLex) (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAC-R) has an important functional role in defining the invasion and metastasis of human colorectal carcinoma. The results were derived from the clinical specimens obtained at surgery or experimental metastasis of human colon carcinoma variant expressing different levels of sLex in nude mice. In the present study, we immunohistochemically examined 132 human colorectal carcinomas for the expression of sLex to investigate whether this antigen expression could serve as a prognostic parameter. The tumors were divided into two groups: sLex positive and sLex negative. The incidence of sLex positive was correlated with the depth of tumor invasion, the presence of the lymph node metastasis, lymphatic invasion, and the disease stage. The difference was statistically significant (P = 0.0026; P = 0.0002; P = 0.003; P = 0.0013; respectively). Based on the data on 114 patients who underwent curative resections, incidence of the disease recurrence was assessed. The sLex-positive patients had higher incidence of recurrence in distant organs, especially in the liver, than that of the sLex-negative patients. The 5-year disease free survival rates of sLex-positive and -negative patients were 57.7 and 89.1%, respectively (P = 0.0002). The difference of 5-year overall survival rates between the two were also significant (sLex positive, 58.3%; sLex negative, 93.0%: P < 0.0001). By Cox multivariate analysis, sLex expression levels remained the best discriminant of disease-free survival (P = 0.035) and overall survival (P = 0.0081). These results suggest that increased expression of sLex is correlated with the extent of malignancy and high incidence of recurrence and consequently with survival of colorectal carcinoma patients. Thus sLex may prove to be a potent marker of recurrence in colorectal carcinoma patients.

Isolation of murine and human homologues of the fission-yeast dis3+ gene encoding a mitotic-control protein and its overexpression in cancer cells with progressive phenotype.

To investigate genes involved in metastasis, we used a differential display method to compare the levels of gene expression in three cell lines derived from murine colon-adenocarcinoma 26 that show different metastatic potentials. The results, and subsequent Northern analyses, confirmed that one gene was expressed most strongly in NL17, the cell line with the highest experimentally metastatic potential to the lung; strongly in NL22, the line with moderately metastatic potential; and very weakly in NL4, which has no metastatic potential in recipient mice. Using this fragment as a probe, we isolated the murine cDNA as well as its human homologue and determined their DNA sequences. The cDNA sequences from both species contained open reading frames of 2874 nucleotides, encoding peptides of 958 amino acids with calculated molecular weights of approximately 109,000; the murine and human nucleotide sequences were 90% identical. The deduced amino acid sequences of these cDNAs revealed significant homology (45% identity) to the dis3+ gene product of Schizosaccharomyces pombe, a protein thought to be essential for mitotic control in the yeast. We therefore termed the murine and human genes hmc (homologue to the mitotic-control gene) and HMC, respectively. In 7 of 13 patients with colorectal cancers and liver metastases, expression of HMC was increased up to 38-fold in primary tumors and metastatic foci as compared to adjacent normal colorectal mucosa. An increase in expression of HMC, its novel product likely to belong to a structurally distinct family of mitotic-control proteins, may be associated with malignant phenotypes of some colorectal cancers.

Absence of genetic alteration at codon 531 of the human c-src gene in 479 advanced colorectal cancers from Japanese and Caucasian patients.

Activation of c-src, a cellular human gene homologous in sequence to the v-src gene of Rous sarcoma virus, had been thought to play an important role in the progression of several types of human cancers, without having undergone any genetic changes. However, recently truncating mutations at codon 531 of the c-src gene were reported in 12% of the advanced colon cancers, and it was also demonstrated that this change was activating, transforming, tumorigenic, and metastasis promoting. To investigate whether the codon 531-specific mutation could be involved in the carcinogenesis of colorectal cancer in the Japanese and Caucasian populations, we examined a total of 479 advanced colorectal cancers from 421 Japanese patients (46 of them with liver or lung metastases) and from 58 Caucasian patients (11 of them with liver metastases). Using the PCR-RFLP assay and additional single-strand conformation polymorphism analysis, we detected no genetic alteration in any of the advanced colorectal cancers. Our results suggest that the codon 531-specific mutational activation of c-src is unlikely to play a significant role in the malignant progression of colorectal cancers among most Japanese and Caucasian patients.

Activation of the beta-catenin gene by interstitial deletions involving exon 3 in primary colorectal carcinomas without adenomatous polyposis coli mutations.

Among 222 primary colorectal cancers we examined, 58 showed no detectable APC mutations by the protein truncation test. We screened those 58 tumors for somatic mutations in the beta-catenin gene. Although amino acid substitutions in serine or threonine residues in exon 3 had been reported, we found no such mutations; however, in seven tumors, we detected somatic interstitial deletions of 234-760 bp, each of which included all or part of exon 3. Short nucleotide sequences at both ends of each deletion were either identical or complementary, indicating that repeated or inversely repeated sequences were involved in the somatic rearrangements. Reverse transcription-PCR experiments using RNAs isolated from three of these seven tumors detected transcripts that lacked exon 3, in addition to the normal transcript. In one of these cases, we confirmed accumulation of aberrant beta-catenin protein in cytoplasm and nuclei of cancer cells by Western and immunohistochemical analyses. This result suggested that, in the absence of a peptide encoded by exon 3, beta-catenin is stabilized and has a dominant oncogenic effect on colorectal tumorigenesis.

Expression of carbohydrate antigen sialyl Le(a): a new functional prognostic factor in gastric cancer.

PURPOSE: The prognostic value of the altered expression of carbohydrate antigens sialyl Le(a) (sLe(a)) and sialyl Le(x) (sLe(x)), which have been implicated as functional ligands in heterotypic-cell-adhesion systems in the multistep process of tumor metastasis, were evaluated. PATIENTS AND METHODS: The level of expression of sLe(a) and sLe(x) antigens was examined immunohistochemically in paraffin-embedded tumor samples from 137 patients who underwent resection for gastric cancer. Correlation between the antigens' expression, various established clinicopathologic factors, and prognosis were studied by univariate and multivariate analysis. RESULTS: Tumors that were positive for the sLe(a) antigen were significantly more likely to be large (P = .035), to be localized at the proximal third of the stomach (P = .018), to have an infiltrate appearance (P = .013), to have an invasive mode both in depth of invasion (P = .028) and in lymphatic invasion (P = .002), and to be classified as late stage (P = .011) than those that were negative for sLe(a), whereas the sLe(x) antigen status was not correlated with any clinicopathologic factors. The overall survival of patients with an sLe(a)-antigen-positive tumor was significantly poorer than that of those with an sLe(a)-antigen-negative tumor (P = .0001). Survival within each pathologic stage differed also (stage I, P = .030; stage II, P = .046; stage III, P = .026, respectively). A Cox regression analysis with multiple covariates showed that positive sLe(a) antigen status was an independent prognostic factor for a worse outcome in patients with gastric cancer. According to the mode of recurrence, increased sLe(a) antigen expression significantly affected both peritoneal dissemination and liver metastasis. CONCLUSION: Increased expression of the sLe(a) antigen may serve as a potent prognostic indicator for recurrence in patients with gastric cancer. Careful follow-up and intensive therapy are required for patients with an sLe(a)-antigen-positive gastric cancer.

Augmentation of antitumor activity of 5-fluorouracil by interferon alpha is associated with up-regulation of p27Kip1 in human hepatocellular carcinoma cells.

Several clinical trials have demonstrated the effectiveness of combination therapy with 5-fluorouracil (5-FU) and IFN-alpha in colon cancer, hepatocellular carcinoma (HCC), and other malignancies. In our preliminary clinical studies, we have observed outstanding effects with this combination therapy in patients with advanced HCC. However, the underlying mechanism by which IFN-alpha modulates the effects of 5-FU is unknown. We, therefore, conducted a mechanistic study using two HCC cell lines, PLC/PRF/5 and HuH7. IFN-alpha significantly enhanced the growth inhibitory effect of 5-FU in PLC/PRF/5 cells but not in HuH7 cells, and the isobolographic analysis indicated that this effect was synergistic. Flow cytometric analysis showed a delay in the progression of G0-G1 to S phase in PLC/PRF/5, and a sustained, induction of the cyclin-dependent kinase inhibitor p27-Kip1 and down-regulation of cyclin D1 was observed. Moreover, increased expression of p27Kip1 was associated with reduced CDK-2-associated kinase activity. Another difference in the two cell types was that PLC/PRF/5 expressed abundant IFN receptors, but HuH7 did not. Apoptosis assays were not helpful in explaining the mechanism. Our results suggest that the synergistic effects of 5-FU and IFN-alpha may in part be attributable to alterations in cell cycle progression via up-regulation of p27Kip1.

Genetic detection for micrometastasis in lymph node of biliary tract carcinoma.

The presence of regional lymph node metastasis is one of the most significant poor-prognosis factors in patients with biliary tract carcinoma. To establish a sensitive reverse transcription (RT)-PCR assay to detect micrometastases in lymph nodes of biliary tract carcinoma, we first investigated the optimal markers in biliary tract carcinoma. The expressions of the six candidates for a suitable RT-PCR marker [mammaglobin B, carcinoembryonic antigen (CEA), cytokeratin (CK) 20, prostate-specific antigen, and melanoma antigens (MAGE-1 and MAGE-3)] were evaluated in two bile duct cancer cell lines and human biliary tract carcinoma tissues. Of 32 carcinoma tissues, mammaglobin B, CEA, prostate-specific antigen, MAGE-1, MAGE-3, and CK 20 were expressed in 28 (88%), 26 (81%), 4 (13%), 5 (16%), 7 (22%), and 9 (28%), respectively. Mammaglobin B and CEA were considered to be good markers of the six candidates. We then examined 209 lymph nodes obtained from 15 patients with biliary tract carcinoma by RT-PCR assay using both mammaglobin B and CEA and compared the results with those of histological examination. All of 20 histologically positive lymph nodes for metastasis displayed the PCR product(s) of marker genes. Of 189 histologically negative nodes, 24 (13%) nodes expressed mammaglobin B and/or CEA mRNA, suggesting the presence of micrometastasis. Our findings suggest that mammaglobin B and CEA could be useful RT-PCR markers for the detection of lymph node micrometastases in biliary tract carcinomas. Our RT-PCR assay allows accurate clinical staging necessary for patient stratification with respect to adjuvant therapy after surgery.

Detection of K-ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: correlation with clinicopathological features.

We investigated the presence of K-ras gene mutation in plasma DNA and assessed its clinical value in patients with pancreatic adenocarcinoma. Mutations in codon 12 of the K-ras gene were examined by mutant allele-specific amplification method using DNA extracted from surgical specimens and plasma samples of 21 patients with pancreatic adenocarcinoma. K-ras gene mutation was detected in 15 of 21 (71%) primary tumors. In 9 of 15 (60%) patients with K-ras gene mutation-positive tumors, an identical mutation was detected in the plasma DNA. None of four patients with chronic pancreatitis or five healthy subjects had such mutations in plasma DNA. Tumors positive for K-ras gene mutation in plasma DNA were significantly larger (P = 0.04) and less likely to result in a curative cure after surgical resection (P = 0.09) than those negative for the mutation. Other clinicopathological features, including age, sex, histological type, mode of invasion, and metastasis, did not correlate with K-ras gene mutations in plasma DNA. Treatment resulted in disappearance of K-ras gene mutations in plasma DNA in six of nine (67%) patients. Three patients with a persistently positive K-ras gene mutation in pre- and post-treatment plasma samples were likely to show early recurrence or have a progressive disease. Our findings suggest that K-ras gene mutation can be detected in plasma DNA of patients with pancreatic adenocarcinoma. Detection of K-ras mutations in plasma may be clinically useful for evaluating tumor burden and efficacy of treatment.

Overexpression of cyclooxygenase-2 in carcinoma of the pancreas.

The level of cyclooxygenase (COX)-2 has been investigated recently in various human carcinomas. In the present study, we examined the distribution and extent of COX-2 protein in human pancreatic tumors using immunohistochemistry. A strong expression of COX-2 protein was present in 23 of 52 (44%) pancreatic carcinomas, a moderate expression was present in 24 of 52 (46%) pancreatic carcinomas, and a weak expression was present in 5 of 52 (10%) pancreatic carcinomas. In contrast, benign tumors showed weak expression or no expression of COX-2, and only islet cells displayed COX-2 expression in normal pancreatic tissues. Overexpression of COX-2 in carcinoma tissues was also confirmed by Western blot analysis. Furthermore, consistent with the results at protein levels, reverse transcription-PCR analyses indicated that COX-2 mRNA was overexpressed in 7 of 13 (54%) carcinomas, but in none of 3 benign tumors. Our findings suggest that COX-2 inhibitors might be potentially effective against pancreatic carcinomas and that COX-2 may be involved in certain biological processes in pancreatic islets.

Increased expression of COX-2 in nontumor liver tissue is associated with shorter disease-free survival in patients with hepatocellular carcinoma.

Recent studies have shown increased levels of cyclooxygenase-2 (COX-2) in a variety of human malignancies including hepatocellular carcinoma (HCC), but little is known about the prognostic value of COX-2 in HCC or its associated nontumor liver tissue. We examined the expression of COX-2 protein by immunohistochemistry in 53 patients with HCCs whose corresponding nontumor tissues were hepatitis C virus-related chronic hepatitis (n = 21) and cirrhosis (n = 32). Samples of nine histologically normal livers and eight precancerous dysplasias were also analyzed. The level of COX-2 increased from normal liver to chronic hepatitis to cirrhosis. The majority of cirrhotic livers (81%) displayed marked COX-2 expression. In dysplasias, COX-2 expression was mainly moderate or strong (88%). In HCC, 17% of samples displayed a high COX-2 expression, and 37% of samples expressed COX-2 at a moderate level. Concordant results were obtained with reverse transcription-PCR and Western blot analyses. Clinicopathological survey indicated a significant correlation between COX-2 expression and differentiated carcinoma (P = 0.019). Although there was no correlation between COX-2 expression in HCC and prognosis, a striking difference was found between COX-2 expression in nontumor tissue and shorter disease-free survival (P = 0.0132). Moreover, high COX-2 expression in nontumor tissue was significantly correlated with the presence of active inflammation (P < 0.0001). The present findings suggest that COX-2 expression in nontumor tissue may play a positive role in relapse of HCC after surgery.

Novel host-vector system for selection and maintenance of plasmid-bearing, streptomycin-dependent Escherichia coli cells in antibiotic-free media.

A novel system for selection and maintenance of cells carrying a recombinant plasmid has been developed, using the streptomycin-dependent (Smd) Escherichia coli 4D host and a plasmid vector carrying an rpsL gene from an Sm-resistant (Smr) mutant of E. coli which masks the Smd phenotype. Strain 4D carrying the Smr pBR322 plasmid can grow without Sm. Using this host-vector system, we can select for cells carrying an Smr recombinant plasmid and maintain them in antibiotic-free media.

Construction of novel shuttle vectors and a cosmid vector for the glutamic acid-producing bacteria Brevibacterium lactofermentum and Corynebacterium glutamicum.

Novel cloning vectors for glutamic acid-producing bacteria have been constructed. Two cryptic plasmids, pAM330 from Brevibacterium lactofermentum and pHM1519 from Corynebacterium glutamicum, were used as precursors, and recombined with pBR325 or pUB110. Resultant composite plasmids were able to propagate and to express the CmR or KmR phenotype in B. lactofermentum and C. glutamicum. A smaller, high-copy-number plasmid, pAJ43, was also isolated following deletion of a part of the pAM330-pBR325 composite plasmid. Furthermore, a cosmid vector, which can be packaged and transduced through phage infection, has been developed using a cohesive-end fragment of the f1A phage and plasmid pAJ43. These plasmids are suitable for use as cloning vectors in the glutamic acid-producing bacteria.