Prognostic significance of thymidylate synthase (TS) expression in cutaneous malignant melanoma.

Thymidylate synthase (TS) plays an essential role in the pathogenesis and development of cancer, and TS-targeting agents have been widely used against different types of cancers. However, it remains still unclear whether or not TS is expressed in malignant melanoma. We conducted the clinicopathological study to investigate the prognostic significance of TS expression in cutaneous malignant melanoma. Ninety-nine patients with surgically resected cutaneous malignant melanoma were assessed. Tumor sections were stained by immunohistochemistry for TS, Ki-67, and microvessel density (MVD) determined by CD34. TS was positively expressed in 26% (26 out of 99). The expression of TS was significantly associated with T factor, cell proliferation (Ki-67) and MVD (CD34). By Spearman's rank test, TS expression was significantly correlated with Ki67 and CD34. By univariate analysis, ulceration, disease stage, TS, Ki-67 and CD34 had a significant relationship with survival. Multivariate analysis confirmed that TS was an independent prognostic factor for poor prognosis of cutaneous malignant melanoma. The positive expression of TS could be a useful marker for predicting poor prognosis in patients with cutaneous malignant melanoma, and TS-targeting agents may be worth trying for the treatment of this dismal disease.

Expression of ER stress markers (GRP78/BiP and PERK) in patients with tongue cancer.

The glucose-regulated protein (GRP78/BiP) and PKR-like endoplasmic reticulum kinase (PERK) plays a crucial role in the endoplasmic reticulum (ER) stress response. GRP78/BiP is highly elevated in various human cancers. Our study is to examine the clinicopathological significance of GRP78/BiP and PERK expression in patients with tongue cancer. A total of 85 tongue cancer patients were analyzed, and tumor specimens were stained by immunohistochemistry for GRP78/BiP, PERK, GLUT1, Ki-67 and microvessel density (MVD) determined by CD34.GRP78/BiP and PERK were highly expressed in 47% and 35% of all patients, respectively. GRP78/BiP disclosed a significant relationship with PERK expression, lymphatic permeation, vascular invasion, glucose metabolism and cell proliferation. The expression of GRP78/BiP was significantly higher in metastatic sites than in primary sites (79% vs. 47%, p=0.003). We found that the high expression of GRP78/BiP was proven to be an independent prognostic factor for predicting poor outcome in patients with tongue cancer. In the analysis of PFS, PERK was identified as an independent predictor. The increased GRP78/BiP expression was clarified as an independent prognostic marker for predicting worse outcome. Our study suggests that the expression of GRP78/BiP as ER stress marker is important in the pathogenesis and development of tongue cancer.

Prognostic significance of GRP78/BiP expression in patients with Stage III/IV hypopharyngeal squamous cell carcinoma.

The immunoglobulin heavy chain binding protein (BiP)/glucose-regulated protein 78 (GRP78) plays an essential role in the endoplasmic reticulum (ER) stress, and GRP78/BiP is known to be highly expressed in various human neoplasms. The clinicopathological features of GRP78/BiP expression in patients with advanced hypopharyngeal squamous cell carcinoma (HSCC) remain unclear. The aim of this study is to elucidate the prognostic significance of GRP78/BiP for HSCC.A total of 68 patients with advanced HSCC (stage III/IV) were analyzed, and tumor specimens were stained with immunohistochemistry for GRP78/BiP, Ki-67, and microvessel density (MVD), as determined through CD34 and p53 levels. GRP78/BiP was highly expressed in 80.8% (55/68) of all patients. The expression level of GRP78/BiP disclosed no significant relationship with any variables. Multivariate analysis confirmed that low expression of GRP78/BiP was an independent prognostic factor for predicting poor overall survival and progression-free survival in patients with advanced HSCC. The decreasing expression of GRP78/BiP was identified as a significant predictor related to shorter survival duration after surgery for advanced HSCC. Our study suggests that the reduced expression of GRP78/BiP contributes to worse survival for patients with advanced head and neck cancer.

Biological significance of fluorine-18-α-methyltyrosine (FAMT) uptake on PET in patients with oesophageal cancer.

PURPOSE: (18)F-FAMT as an amino-acid tracer for positron emission tomography (PET) is useful for detecting human neoplasms. (18)F-FAMT is accumulated in tumour cells solely via L-type amino-acid transporter 1 (LAT1). This study was conducted to investigate the biological significance of (18)F-FAMT uptake in patients with oesophageal cancer. METHODS: From April 2008 to December 2011, 42 patients with oesophageal cancer underwent both (18)F-FAMT PET/CT and (18)F-FDG PET/CT before surgical treatment. The immunohistochemical analysis of LAT1, CD98, Ki-67, CD34, p53, p-Akt and p-mTOR was performed on the primary lesions. In vitro experiments were performed to examine the mechanism of (18)F-FAMT uptake. RESULTS: High uptake of (18)F-FAMT was significantly associated with advanced stage, lymph node metastasis and the expression of LAT1, CD98, Ki-67 and CD34. LAT1 expression yielded a statistically significant correlation with CD98 expression, cell proliferation, angiogenesis and glucose metabolism. In vitro experiments revealed that (18)F-FAMT was specifically transported by LAT1. CONCLUSIONS: The uptake of (18)F-FAMT within tumour cells is determined by the LAT1 expression and correlated with cell proliferation and angiogenesis in oesophageal cancer. The present experiments also confirmed the presence of LAT1 as an underlying mechanism of (18)F-FAMT accumulation.

Phase II multi-institutional prospective randomised trial comparing S-1+paclitaxel with S-1+cisplatin in patients with unresectable and/or recurrent advanced gastric cancer.

BACKGROUND: A combination of S-1 and cisplatin has been shown to be effective with acceptable safety for the first-line treatment of far-advanced gastric cancer in Japan. This is the first randomised phase II trial to compare S-1+paclitaxel with S-1+cisplatin in this setting. METHODS: Patients with unresectable and/or recurrent advanced gastric cancer were randomly assigned to receive one of the two regimens: S-1 (40 mg m(-2) twice daily) on days 1-14 plus paclitaxel (60 mg m(-2)) on days 1, 8, and 15 of a 4-week cycle (S-1+paclitaxel) or S-1 (40 mg m(-2) twice daily) on days 1-21 plus cisplatin (60 mg m(-2)) on day 8 of a 5-week cycle (S-1+cisplatin). The primary end point was the response rate (RR). Secondary end points included progression-free survival (PFS), overall survival (OS), and safety. RESULTS: A total of 83 patients were eligible for safety and efficacy analyses. In the S-1+paclitaxel and S-1+cisplatin groups, RRs (52.3% vs 48.7%; P=0.74) and median PFS (9 vs 6 months; P=0.50) were similar. The median OS was similar in the S-1+paclitaxel and S-1+cisplatin groups (16 vs 17 months; P=0.84). The incidence of grade 3 or higher haematological toxicity was 19.0% with S-1+paclitaxel and 19.5% with S-1+cisplatin. The incidence of grade 3 or higher non-haematological toxicity was 14.2% with S-1+paclitaxel and 17.1% with S-1+cisplatin. CONCLUSION: S-1+paclitaxel was suggested to be a feasible and effective non-platinum-based regimen for chemotherapy in patients with advanced gastric cancer. Our results should be confirmed in multicenter, phase III-controlled clinical trials.

The peptide hormone xenin induces gallbladder contractions in conscious dogs.

Xenin is a 25-amino acid peptide isolated from human gastric mucosa. The biological activities of xenin include modulating intestinal motility and affecting exocrine pancreatic secretion and gastric acid secretion. The physiological effect of xenin on the gastrointestinal tract, however, is incomplete. The objective of this study is to investigate the effects of xenin on the gastrointestinal tract motility of conscious dogs. Gastrointestinal tract and gallbladder contractions were monitored by chronically implanted force transducers. Synthetic xenin was injected intravenously during the interdigestive state with or without pretreatment with cholinergic blockers. The effects of xenin following cholecystectomy and truncal vagotomy were also investigated. Xenin induced gallbladder and jejunal contractions, although a dose-dependent response was shown only with gallbladder contractions. These effects were inhibited by pretreatment with cholinergic blockers, but were not enhanced by truncal vagotomy. The jejunal contractions were completely inhibited by cholecystectomy. The only direct effect of xenin in terms of gastrointestinal motility was to induce gallbladder contractions in conscious dogs. The neural pathway mediating xenin's action was cholinergic, but not the vagal. This novel finding indicates a new role of xenin.

Experimental results and early clinical experience with an easy method for intracorporeal knot tying using a novel laparoscopic needleholder.

BACKGROUND: Intracorporeal suturing and knot tying are among the most difficult procedures in laparoscopic operations. An easy and inexpensive method for intracorporeal instrumental ligation with a modified laparoscopic needle driver is presented. METHODS: The needle driver developed in this study has a novel mechanism that can fix the suturing thread in a hook at the distal site of the holder's jaw hinge. This hook projects out from the rod only when the jaw of the holder is open. After the needle is removed from the tissue using the grasper, the needle driver is placed under the grasper, which the surgeon manipulates by the left hand. Then the thread is hooked on the needle driver by withdrawal of the driver with the jaw opening. The tip of the needle driver is moved over the shaft of the grasper by keeping the thread on the hook. The thread is entwined during a series of crossing movements of the rods of the forceps. The short tail of the suture material is gripped and tied up as a first throw of ligation. The side edge of the jaw, used for thread cutting, is sharpened by grinding. RESULTS: When the angle of the forceps is set at 90 degrees in the box trainer, no difference in terms of ligation time and degree of error is observed between the hook and conventional C-loop methods. In the case of the 30 degree forceps angle, the novel method is superior to the conventional method. CONCLUSION: The novel needle driver provides an easy and inexpensive method for performing an intracorporeal ligation, particularly in a case involving a sharp axis angle of the forceps. More clinical experience is necessary for evaluation of this method, but it has potential advantages in laparoscopic operations.

Ghrelin does not stimulate gastrointestinal motility and gastric emptying: an experimental study of conscious dogs.

Ghrelin is a peptide that was discovered in endocrine cells of the stomach. However, its action in regulating the fasted and fed motor activity of the digestive tract is not fully understood. In the present study, we examined the effects of an intravenous (i.v.) injection of canine ghrelin on the physiological fasted and fed motor activities in the stomach, duodenum, jejunum and colon of freely moving conscious dogs. An i.v. injection of canine ghrelin released growth hormone in a dose-dependent manner; however, it did not stimulate the motor activity of the digestive tract in either the fasted or the fed state. Moreover, an i.v. injection of high-dose canine ghrelin significantly reduced the motility index in the gastric body in the fasted state. Ghrelin did not accelerate gastric emptying, either. These results differ from previous reports dealing with rodents. It is significant that such results were obtained in research with dogs, which are larger animals.

Eradication of hepatic metastases of carcinoma H-59 by combination chemoimmunotherapy with liposomal muramyl tripeptide, 5-fluorouracil, and leucovorin.

We have investigated the effect of a combined chemoimmunotherapy protocol with liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE), 5-fluorouracil (5-FU), and 5-formyltetrahydrofolate (leucovorin) on the growth of hepatic metastases using carcinoma H-59, a liver-homing subline of the Lewis lung carcinoma (P. Brodt, Cancer Res., 46: 2442-2448, 1986). C57BL/6 mice inoculated with the tumor cells via the intrasplenic route received three i.v. injections of liposomal MTP-PE, the first of which was administered 3 days prior to tumor cell inoculation. Chemotherapy with 5-FU and leucovorin at the maximal tolerated doses (30 mg/kg per injection) was initiated immediately after tumor inoculation and continued on alternate days for a total of 4 injections. The incidence of liver metastases in animals which received the combined therapy was compared to that in animals treated with chemotherapy or immunotherapy alone. We found that while the number of liver metastases was reduced in all of the treatment groups as compared to control untreated or placebo-treated animals, the combined effect of 5-FU leucovorin and liposomal MTP-PE was significantly better than that of chemotherapy or immunotherapy alone. This was reflected in a reduced incidence (70% as compared to 100% in all other groups) and in a significant reduction in the number and size of the liver nodules. Our results suggest that the efficacy of 5-FU and leucovorin in the treatment of hepatic metastases could be significantly augmented by the addition of the liposome-encapsulated immunoadjuvant MTP-PE.

Genetic and enzymatic evidence for Lewis enzyme expression in Lewis-negative cancer patients.

It has been observed that the frequency of individuals with Lewis-negative erythrocytes is significantly higher in cancer patients than in healthy controls. In this study, 20 of the 66 (30.3%) patients with various cancers were typed as Lewis negative from their erythrocytes, while the same frequency in healthy controls was 11.1%. These 20 patients were divided into three groups based on the presence of Lewis blood group antigens and alpha 1-->4-fucosyltransferase in their salivas: group I, 6 patients who had both Lewis antigens and alpha 1-->4-fucosyltransferase activity; group II, 8 patients who had no Lewis antigens but possessed alpha 1-->4-fucosyltransferase activity; group III, 6 patients who had neither Lewis antigens nor alpha 1-->4-fucosyltransferase activity. The genotyping of Le genes by the PCR-RFLP methods, which have been developed and established by us recently, demonstrated that all 14 patients from groups I and II possess Le gene homozygously (Le/Le) or heterozygously (Le/le), whereas all 6 patients from group III were le/le homozygotes. Only the 6 patients from group III were identified as the genuine Lewis-negative individuals. The immunohistochemical staining of the colorectal tumors also showed that the Lewis antigens could be detected on the tumors from groups I and II but not from group III.

Interaction of second and third domains of Japanese quail ovomucoid with ten mammalian trypsins.

Second and third domains were prepared from Japanese quail ovomucoid and association equilibrium constants, Kas, were measured at 25 degreesC and pH 8 for these domains with trypsins from ten mammalian species: cat, cow, dog, guinea pig, hog, horse, man, rabbit, rat, and sheep. The values ranged from 108 M-1 to 1010 M-1 for the second domain-trypsin associations and from 106 M-1 to 108 M-1 for the third domain-trypsin associations. Changes in Ka values for the interactions between the trypsins and each domain are attributed to slight changes in surface conformation caused by the residue changes in the inhibitor-binding region other than the S1 pocket of the trypsin species. The representative of such residue changes is assumed to be the one observed at residue 217 of trypsin molecule. Concerning each trypsin, the Ka value with the second domain was always higher than that with the third domain. However, the ratios between the two equilibrium constants varied from 3 to 60 depending upon trypsin species. This means that amino acid changes in enzyme-contact residues other than the P1 site of the Kazal-type inhibitor can make it possible to recognize even a slight difference in inhibitor-binding surface among the enzymes with the same S1 pocket and highly similar overall three-dimensional structure.

Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo.

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer-graphic modeling based on the stereo-structure. The common fragment, N-(L-trans-carbamoyloxyrane-2-carbonyl)-phenylalanine-dimethyla mide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10(-7) M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10(-7) M in vitro.

Tumor cells as the origin of elevated serum alpha1,3fucosyltransferase in association with malignancy.

We have previously reported that the elevated activities of serum alpha 1,3fucosyltransferase reverted to normal levels after curative removal of the tumors. To determine the origin of elevated serum alpha 1,3fucosyltransferase, blood samples were obtained from both the drainage vein and the artery in patients with different stages of colorectal cancer at surgery. The enzyme levels in all samples from the drainage vein were found to be higher than the levels in the artery that fed the tumor. Hence, the origin of elevated alpha1,3fucosyltransferase in serum was thought to be the tumor rather than the liver that is the normal source of serum alpha1,3fucosyltransferase. When serum samples not only from colorectal cancer patients but also from patients with gastric, liver, lung, pancreas, bladder and esophagus cancer were treated with anti-FUTVI antibody, the measured activities of alpha1,3fucosyltransferase were markedly reduced. Further, secretion of alpha1,3fucosyltransferase from human colorectal carcinoma cells was also detected in the culture medium by Western immuno-blot analysis with anti-FUTVI antibody.

A new method for assaying adhesion of cancer cells to the greater omentum and its application for evaluating anti-adhesion activities of chemically synthesized oligosaccharides.

A new ex vivo method for assaying adhesion of cancer cells to the greater omentum has been developed using mouse greater omentum and [3H]labelled human gastric and mouse colorectal cancer cells. Since the adhesion rates were found to increase up to 18 h and labelled cells seemed to be stable during the period, the present method could be useful for investigating adhesion of cancer cells to the greater omentum, which must occur at the first step of the peritoneal dissemination. The adhesion of cancer cells to the greater omentum was inhibited by a series of chemically synthesized oligosaccharides and Gal beta1,3[3OMeGal beta1,4GlcNAc beta1,6]alphaBn was found to be the best inhibitor. The anti-tumor effect of this novel tetrasaccharide in vivo was shown in preliminary experiments using Balb/c mice and colon26 cells.

TAT-59, a new triphenylethylene derivative with antitumor activity against hormone-dependent tumors.

The antiestrogenic action of TAT-59 [(E)-4-[1-[4-[2-(dimethylamino)ethoxy]-phenyl]-2-(4-isopropyl) phenyl-1-butenyl]phenyl monophosphate] was characterized and compared with that of Tamoxifen (TAM). Its active metabolite, 4-OH-TAT-59, had a high binding affinity to estrogen receptor (ER), present in the cytosol of the uterus of immature rat, similar to estradiol. TAT-59 and 4-OH-TAT-59 inhibited in vitro estrogen-stimulated proliferation of MCF-7 cells at a lower concentration than TAM. In the absence of estradiol, TAT-59 and 4-OH-TAT-59 were effective at a lower concentration than that of 4-OH-Tamoxifen (4-OH-TAM), the active metabolite of TAM. In uterine growth inhibition, the effective dose of TAT-59 was about 3-6-fold lower than that of TAM, in various administration schedules. The minimum effective dose of TAT-59 against in vivo MCF-7 cells was about 3-fold lower than that of TAM. In DMBA-induced rat mammary tumors, TAT-59 inhibited the growth of existing tumors at about a 10-fold lower dose than TAM. Especially in the tumors with low ER levels (10-20 fmol/mg protein), TAT-59 showed a significantly stronger inhibitory effect than TAM. These experiments showed that TAT-59 was more effective in lower doses than TAM, even against the tumors with low ER content.

Semiquantitative SPECT tumor uptake of technetium-99m-labeled anti-CEA monoclonal antibody in colorectal tumor.

UNLABELLED: Technetium-99m-BW431/26 images were compared to images of resected tumor specimens to evaluate the efficacy and accuracy of SPECT imaging of colorectal carcinoma. METHODS: Immunoscintigraphy with 99mTc-BW431/26 was performed on seven patients with colorectal carcinoma and one patient with a benign colorectal tumor. Accumulation of 99mTc-BW431/26 in the tumor was expressed as the tumor-to-normal tissue count ratio (SPECT T/N ratio) calculated by a region of interest method on the SPECT images obtained 24 hr after administration of 99mTc-BW431/26. All patients underwent research of the tumor immediately after 24-hr imaging, and the radioactivity in tumor specimen and normal tissue was measured to calculate the tissue T/N ratio. RESULTS: SPECT demonstrated definite increased tracer uptake by the tumor in all colorectal cancer patients. The benign lesion showed tracer uptake to a lesser extent. SPECT, however, failed to visualize a 10-mm lesion in a patient with familial adenomatous polyposis despite a tissue T/N ratio of 4.8, while autoradiography showed radioactivity uptake in the polyps. CONCLUSION: Although SPECT has limitations in detecting small lesions because of its limited spatial resolution, T/N ratios could be measured exactly by SPECT if the lesion is of a certain volume. SPECT imaging with 99mTc-BW431/26 can precisely evaluate tracer uptake in tumors and predict the efficacy of radioimmunotherapy in patients with colorectal cancer.

CEA levels in peritoneal washings from gastric cancer patients as a prognostic guide.

Carcinoembryonic antigen (CEA) levels were determined in the peritoneal washings from 44 patients with gastric cancer to evaluate the usefulness for a predictor of postsurgical prognosis. Seventeen of the 21 patients (80.9%) with serosal invasion showed elevated levels of CEA, whereas most of the patients with no serosal invasion (22/23) showed low levels of CEA and did not develop peritoneal metastasis. All patients with positive cytology showed elevated levels of CEA in the peritoneal washings. Therefore, CEA levels in the peritoneal washings could be an adjunctive tool for predicting the postsurgical prognosis in gastric cancer.

A novel ex vivo method for assaying adhesion of cancer cells to the peritoneum.

A novel ex vivo method to determine the cell adhesion of cancer cells to the peritoneum was described. The wells of a microtiter plate were filled with cell suspension and sealed using mouse peritoneum. The peritoneum was fixed using a plastic cover and the plate was turned upside down and incubated for cell adhesion. After incubation for 80 min, the plate was centrifuged and non-adherent cells were assayed by MTT assay. Human cancer cells (MKN28, MKN45, MKN74, KM12C and KM12SM) adhered to the mouse peritoneum as well as cells from mouse (Colon26) and the ratio of cells attached to the peritoneum was estimated to be between 12.8 and 66.4%. This simple method could be useful to investigate the adhesion molecule associated with peritoneal dissemination.

Animal model of para-aortic lymph node metastasis.

The purpose of this study was to establish a model of experimental lymph node metastasis by intra-rectal implantation of human cancer cells in nude mice. Four types of human cancer cell lines (TE-1, MKN-45, HT-29, and MIAPaca-2) were investigated. Tumor cells suspended in Matrigel were injected into the submucosal layer of the rectum. All cancer cell lines produced locally aggressive rectal tumors and, subsequently, para-aortic lymph node metastasis. We were unable to produce other distant metastases in the dying state in such locations as the liver, spleen, lung, and peritoneum. However, using this method, we were able to evaluate the effect of the anti-cancer agent uracil/tegafur (UFT) on primary tumor growth and lymph node metastasis. Oral intake of UFT significantly suppressed implanted tumor volume and inhibited lymph node metastasis. We expect that the process of lymph node metastasis shown in this model will be studied as an experimental model of lymph node metastasis simulating human cancers.

A novel model for invasion of cancer cells using the submucosal layer of the human stomach.

An in vitro model for studying the invasion mechanism of cancer cells was established using the submucosal layer of the human stomach. Submucosa prepared from a surgical specimen was maintained in an organ culture. The cytoarchitecture of the cultured submucosa remained well preserved; viability remained for over 2 weeks. When human gastric cancer cell lines MKN45, MKN74, and Kato III were seeded onto the submucosal slices, cancer cells of MKN45 and KATO III invaded the submucosa 3 days after inoculation. However, MKN74 cells were not seen in the submucosal slices. Our invasion model, which mimics the in vivo conditions of the submucosa of human stomach, may make it possible to analyze actual events of human gastrointestinal malignant cell invasion in normal submucosa in vitro. The usefulness of our invasion model lies in the choice of the submucosal layer of the human stomach as the host tissue. The histarchitecture of the submucosal slices indicates that the model has potential for studies of the mechanism of interactions between carcinoma cells and host tissue similar to interactions that may occur in vivo. Moreover, this method allows the continuous microscopic observation of cells within the living submucosa. Using this model, a novel approach to controlling the local invasion of tumor cells may lead to a promising, radical cure for these intractable neoplasms. Our model system is an in vitro model that is facile, inexpensive, and experimentally manipulative.