Two cases of BRAF-mutated, bulbar conjunctival melanoma, and review of the published literature.

We describe two patients with BRAF-mutated melanoma of the epithelioid cell type arising from primary acquired melanosis with severe atypia of the right bulbar conjunctiva. Patient 1 was a 71-year-old Japanese man. After adjuvant cryotherapy and enucleation of the right eyeball, therapy with vemurafenib was administered for a distant metastasis to a lumbar vertebra, accompanied by erythema multiforme and two keratinous tumours. The patient died due to metastases to the liver and multiple vertebrae, despite therapy with nivolumab and combination therapy with dabrafenib plus trametinib. Patient 2 was a 72-year-old Japanese man. After adjuvant cryotherapy, periodic mitomycin C eye drops, and excision of the superficial portion of the right parotid gland and the dissection of cervical lymph nodes, he was treated with adjuvant combination therapy with dabrafenib plus trametinib. Dermatologists should be familiar with BRAF-mutated conjunctival melanoma, which is usually located on the bulbar conjunctiva and associated with more frequent distant metastasis.

Spontaneous endometriosis in cynomolgus monkeys as a clinically relevant experimental model.

STUDY QUESTION: Does spontaneous endometriosis in cynomolgus monkeys have the characteristics required of a good experimental model? SUMMARY ANSWER: Spontaneous endometriosis in cynomolgus monkeys exhibited similar clinicopathological characteristics to the human disease and was useful as an experimental model. WHAT IS KNOWN ALREADY: The prevalence of endometriosis in autopsied cynomolgus monkeys (Macaca fascicularis) in a breeding colony was reported to be 28.7% in 1993. The histopathological findings we reported recently showed that components of spontaneous endometriosis were not only endometriotic epithelium and stromal cells (CD10-positive) with hemorrhage and inflammation, but also smooth muscle metaplasia and nerve fibers. STUDY DESIGN, SIZE, DURATION: During routine medical examinations at a research facility from 2008 to 2012, 614 female cynomolgus monkeys of reproductive age (6-25 years) were screened for endometriosis by the presence of regular menstrual bleeding, serum CA125 levels and palpation of the abdomen. In total, 29 monkeys were selected as subjects for the following study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 29 monkeys selected, 15 were diagnosed with endometriosis by laparoscopy and/or open surgery. The monkeys were monitored by observing their general condition, and eight of these were monitored using laparoscopy and MRI. In addition, to investigate appropriate screening parameters and endometriosis-associated biological parameters in monkeys, we retrospectively examined general laboratory parameters that correlate to the menstrual cycle and disease status. MAIN RESULTS AND THE ROLE OF CHANCE: The combination of CA125 serum levels (this was a useful marker for chocolate cysts), palpation of the abdomen, and fecal abnormalities was the most efficient screening method for diagnosing monkeys with endometriosis. Each animal could be diagnosed and assigned a disease stage by laparoscopy. While monitoring the disease stage by laparoscopy and/or MRI, disease status in individual monkeys was mainly stable or was progressive for 2-7 months. The detection rate by screening was low (15/614) but age-specific analysis suggests that screening would be more efficient if a colony for an endometriosis model is maintained with 11-20-year olds. As an endometriosis-associated biological parameter, the decrease in food consumption that coincided with menstruation was selected and correlated well (R2 value = 0.8239) with disease status (according to a modified adhesion revised American Fertility Society score). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Peritoneal fluid was not analyzed because a smaller amount is produced in cynomolgus monkeys than in baboons. Although clinical endometriosis-associated pain is evaluated in women using a visual analog scale, pain could not be directly evaluated in this non-human primate model. WIDER IMPLICATIONS OF THE FINDINGS: Although cynomolgus monkeys are relatively small (2-5 kg) primates, laparoscopy and MRI make it possible to evaluate spontaneous endometriosis in these monkeys and to monitor its development over time. Spontaneous endometriosis in cynomolgus monkeys is a useful model for evaluating disease progress and drug efficacy because they have similar lesions to those in humans, and conventional laboratory methods and parameters for assessment are well established. STUDY FUNDING/COMPETING INTEREST(S): No external funds were used for this study. A.N.-K., K.T., H.T., A.K. and M.S. are full-time employees of Chugai Pharmaceutical Co., Ltd. R.K. received a consultancy fee from Chugai Pharmaceutical Co., Ltd. and lecture fees from Chugai Pharmaceuticals, Japan Vaccine Co. Ltd., Merck & Co., Mochida Co. Ltd., Roche Diagnostics, and BD, unrelated to the submitted work. S.N., S.O. and T.S. have nothing to declare.

Restoration of innate and adaptive immune responses by HCV viral inhibition with an induction approach using natural interferon-beta in chronic hepatitis C.

Chronic hepatitis C (CHC) is a serious medical problem necessitating more effective treatment. This study investigated the hypothesis that an induction approach with nIFN-beta for 24 weeks followed by PEG-IFN-alpha+ribavirin (standard of care: SOC) for 48 weeks (novel combination treatment: NCT) would increase the initial virologic response rate and restore innate and adaptive immune responses in CHC. Seven CHC patients with a high viral load and genotype 1b were treated with NCT. Serum cytokine and chemokine levels were evaluated during NCT. NCT prevented viral escape and breakthrough resulting in persistent viral clearance of HCVRNA. IL-15 was increased at the end of induction therapy in both early virologic responders (EAVRs) and late virologic responders (LAVRs); CXCL-8, CXCL-10, and CCL-4 levels were significantly decreased (P < 0.05) in EAVR but not in LAVR during NCT, and IL-12 increased significantly (P < 0.05) and CXCL-8 decreased significantly (P < 0.05) after the end of NCT in EAVR but not in LAVR. NCT prevented viral breakthrough with viral clearance leading to improvement of innate and adaptive immunity resulting in a sustained virologic response (SVR). NCT (n = 8) achieved a higher SVR rate than SOC (n = 8) in difficult-to-treat CHC patients with genotype 1 and high viral loads.

Carcinoembryonic antigen-specific suicide gene therapy of cytosine deaminase/5-fluorocytosine enhanced by the cre/loxP system in the orthotopic gastric carcinoma model.

Tumor-specific gene delivery is crucial to achieving successful effects in suicide gene therapy. Carcinoembryonic antigen (CEA) promoter has been widely used for this purpose, but the expression level of tumor-specific promoters such as CEA promoter is generally low. In the previous study, we used the Cre/loxP system and showed that LacZ expression by the CEA promoter was remarkably enhanced and maintained its specificity using the Cre/loxP regulation system. In this study, the Cre/loxP system was first applied to augmentation of selective expression of the cytosine deaminase (CD) gene as a suicide gene therapy in CEA-producing cells. The double infection with AxCEANCre expressing Cre recombinase under the control of the CEA promoter and AxCALNLCD expressing the CD gene under the control of the CAG promoter by the Cre switching system rendered CEA-producing tumor cells 13-fold more sensitive to 5-fluorocytosine (5-FC) compared with the single infection with AxCEACD expressing CD gene driven by the CEA promoter. The therapeutic efficacy of the enhanced CD/5-FC suicide gene therapy was evaluated in orthotopic implantation models of human gastric carcinoma. Adenovirus vectors (1 x 10(9) plaque-forming units) were administered i.p. into mice three times, and then 5-FC was administered i.p. for the next 10 days. Tumor volume and weight in mice treated with AxCEANCre and AxCALNLCD/5-FC were significantly reduced as compared with those in mice treated not only with Mock (AxCALacZ) but also with AxCEACD/5-FC (P < 0.0001). This beneficial effect on tumor burden was also reflected in the overall survival. The survival periods of the mice treated with AxCEANCre and AxCALNLCD/5-FC were longer than those of mice treated with Mock or AxCEACD/5-FC (P < 0.01). These results suggested that application of the Cre/loxP system could provide a new approach for enhanced selective suicide gene therapy of CD/5-FC for the treatment of advanced gastric carcinoma.

Enhanced expression of the DNA topoisomerase II gene in response to heat shock stress in human epidermoid cancer KB cells.

Type II DNA topoisomerase breaks both DNA strands, and many anticancer agents including etoposide (VP-16) and teniposide (VM-26) have been developed by targeting topoisomerase II molecules. In this study we examined whether expression of the topoisomerase II gene is regulated in response to heat shock stress in human epidermoid cancer KB cells. Exposure of KB cells to 42 degrees C for 3 to 24 h permitted cell growth at a slightly reduced rate but still at an exponential rate, in comparison with that at 37 degrees C, whereas exposure to 45 degrees C for 15 to 120 min caused the almost complete cessation of exponential growth. There appeared 5-fold or higher increases in mRNA levels of both topoisomerase II and a heat shock protein, hsp-70, after exposure to 42 degrees C for 3 h, but only a slight, if any, increase in topoisomerase I mRNA. Nuclear run-on assays showed increased transcription of topoisomerase II and the hsp-70 gene after exposure to 42 degrees C. By contrast, KB cells induced a rapid and transient increase of topoisomerase II mRNA after exposure to 45 degrees C for 15 to 30 min, whereas the cellular level of hsp-70 mRNA was dramatically enhanced 60 min after exposure to 45 degrees C. The immunoblot assay also demonstrated increased expression of topoisomerase II in KB cells exposed to 42 degrees C. Decatenation activity of the nuclear extracts from KB cells was increased 1.5-fold by exposure to 42 degrees C, but there appeared no increase in topoisomerase I activity. Prior exposure of KB cells to 42 degrees C enhanced the cytotoxicity of VP-16, but not that of a topoisomerase I-targeting agent, a camptothecin analogue, CPT-11. However, exposure of KB cells to 42 degrees C after treatment with VP-16 did not enhance the cytotoxicity induced by the drug. The formation of cleavable DNA-topoisomerase II-VP-16 complexes was also greatly increased by prior exposure to 42 degrees C. Our present study proposes the hypothesis that the topoisomerase II gene might be one of the heat-shock-inducible genes and that hyperthermic anticancer therapy with topoisomerase II-targeting antitumor agents can be improved.

Normotensive primary aldosteronism: report of a case.

A 23-yr-old male patient with normotensive primary aldosteronism is reported. He complained of muscle weakness, polydipsia, and polyuria. His blood pressure was generally 118/60 to 124/70 mm Hg. Serum sodium, potassium and chloride were 152.2.2, and 108 meq/liter, respectively. Arterial blood pH, glomerular filtration rate, renal plasma flow and circulating plasma and blood volumes were normal, and plasma bicarbonate was normal or elevated. PRA was 0.16 ng/ml.h and did not increase significantly after sodium deprivation, ambulation, and iv furosemide injection. Plasma aldosterone was 64.1 ng/100 ml. He showed pressor responses to infused angiotensin II and norepinephrine which were similar to those in normal men. Adrenal scintiscanning after iv injection of [131I]6 beta-iodomethyl-19-nor-cholesterol during dexamethasone administration showed dense uptake on the right adrenal and minimal uptake on the left. Intravenous infusion of angiotensin III at a rate of 20 ng/kg. min for 30 min did not cause an increase in plasma aldosterone. Serum electrolytes became normal after spironolactone but not after dexamethasone. At surgery, the right adrenal, bearing a benign adenoma, was removed. After surgery, blood pressure was unchanged, but all biochemical abnormalities disappeared. The cause of this normotension remains to be elucidated, but the diagnosis criteria of primary aldosteronism should now be partly modified.

Involvement of protein kinase in environmental stress-induced activation of human multidrug resistance 1 (MDR1) gene promoter.

The human MDR1 gene can be induced in response to various environmental stimuli. To examine whether such stress-induced activation of the MDR1 gene can be modulated by protein kinase, we employed a stable human cancer KB cell line which contained the bacterial chloramphenicol acetyltransferase (CAT) gene directed by the MDR1 gene promoter. H-7, a protein kinase C inhibitor, at more than 40 microM inhibited activation of the MDR1 promoter that was induced by ethylmethane sulfonate, 5-fluorouracil or UV irradiation. DNA binding activity of nuclear factors recognizing the MDR1 promoter was augmented in KB cells treated with UV, but decreased in cells treated concomitantly with H-7. Okadaic acid alone was able to induce the promoter activation, and this induction was dependent on specific promoter sequences. Okadaic acid also enhanced the DNA binding activity of nuclear factors recognizing the MDR1 promoter. The phosphorylation of transacting factors may modulate the MDR1 gene promoter activity.

Vitamin K concentrations in the plasma and liver of surgical patients.

Vitamin K deficiency has been reported in patients who were treated with antibiotics and placed on poor diets after surgery. High-performance liquid chromatography (HPLC) was used to study the influence of dietary intake on vitamin K concentrations in surgical patients (n = 22). Plasma phylloquinone decreased rapidly from 1.19 +/- 0.16 to 0.47 +/- 0.12 nmol/L (means +/- SEM, n = 11) on a low-phylloquinone diet and from 1.16 +/- 0.12 to 0.36 +/- 0.07 nmol/L (n = 11) by postoperative fasting. A small amount of phylloquinone and a large amount of menaquinone were found in liver tissue. Phylloquinone concentration was 28.0 +/- 4.3 pmol/g liver (wet weight) on the standard diet (n = 7) whereas it was 6.8 +/- 1.1 pmol/g on the low-phylloquinone diet after 3 d (n = 8). Because phylloquinone is rapidly depleted by fasting, it may be difficult to prevent vitamin K deficiency by dietary phylloquinone alone during long-term fasting after surgery.

Chemosensitivity testing with highly purified fresh human tumour cells with the MTT colorimetric assay.

A major problem associated with the succinate dehydrogenase inhibition (SDI) test using tetrazolium dye (MTT) as a cancer chemosensitivity testing is the contamination of non-malignant cells in the tumour tissues. Highly purified fresh human tumour cells from 44 solid tumours and 24 malignant ascites were used for the MTT assay. The purity of tumour cells was greater than 90% after separation on Ficoll-Hypaque and Percoll discontinuous gradients. The OD570 obtained from tumour cells alone was higher than that from non-malignant cells. The chemosensitivity of tumour cells was distinct from that of non-malignant cells. Moreover, the chemosensitivity of highly purified tumour cells was also distinct from that of non-purified cells just separated from tumour tissues. 31 of the 68 patients had evaluable lesions, and received cancer chemotherapy according to the results of MTT assay using highly purified tumour cells. A clinical response was obtained in 10 of the 31 patients (response rate = 32.3%, 5 complete responses, 5 partial responses).

Synergistic effects of tamoxifen and cepharanthine for circumventing the multidrug resistance.

We examined the synergistic effects of tamoxifen (TAM) and cepharanthine (CEP) for doxorubicin (DOX) sensitivity using MTT assay. The augmentation of DOX sensitivity by TAM and CEP was significantly correlated with the P-glycoprotein expression. The cytotoxic effect of DOX with TAM and CEP was significantly higher than that of DOX alone, or DOX with TAM, and this synergistic effect was dominant in cell lines with high expression of P-glycoprotein. It was also examined that the intracellular concentration of DOX was increased in combined exposure of TAM and CEP, compared with the exposure of TAM, because TAM and CEP promoted the influx and inhibited the efflux of DOX. Thus, TAM and CEP might be able to circumvent DOX-resistance for treatment in cancer patients.

Clinical significance of quantitative analysis of carcinoembryonic antigen assessed by flow cytometry in fresh human gastric cancer cells.

The expression of carcinoembryonic antigen(CEA) on tumor cells freshly excised from 51 patients with gastric cancer was studied using flow cytometry. The expression of CEA by flow cytometry was more quantitative than that by immunohistochemical staining. There was no relationship between the fluorescence intensity assessed by flow cytometry and serum CEA levels, except for patients with a high titer of serum CEA. The patients with high grade CEA expression on tumor cells by flow cytometry had poor prognoses, compared to patients with low CEA expression in undifferentiated gastric cancer. Thus, it is suggested that the quantitative CEA expression on tumor cells by flow cytometry could be a useful prognostic marker in postoperative gastric cancer patients.

Overexpression of multidrug resistance genes MDR1 and cMOAT in human hepatocellular carcinoma and hepatoblastoma cell lines.

Overexpression of either of the multidrug resistance genes MDR1 and MRP is associated with resistance of tumors to multiple chemotherapeutic agents. Overexpression of MDR1 has been reported in some cell lines derived from human hepatocellular carcinomas (HCC) and hepatoblastomas (HB). The human gene for cMOAT (), a homologue of MRP, is thought to mediate hepatobiliary excretion of organic anions and to be associated with cisplatin resistance. In this study, expression levels of MDR1 and cMOAT were examined in 9 human HCC and HB cell lines and 10 other human cancer cell lines. Overexpression of the cMOAT gene was observed in all 9 HCC and HB cell lines and 3 of 10 other cancer cell lines. Co-overexpression of the cMOAT and MDR1 genes was observed in 7 of 9 HCC and HB cell lines, but in none of the 10 other cancer cell lines. Seven of the HCC and HB cell lines that had overexpression of the cMOAT gene were shown to be highly resistant to cisplatin compared to 2 HCC cell lines with low levels of cMOAT expression. These findings suggest that overexpression of cMOAT could contribute to cisplatin resistance in HCC and HB.

Activation of the human multidrug resistance-1 (mdr1) gene promoter in response to inhibitors of DNA topoisomerases.

The multidrug resistance (MDR1) gene encodes a Mr 170,000 energy-dependent membrane efflux pump termed P-glycoprotein, and the P-glycoprotein is often expressed in various human tumors before and after cancer chemotherapy. In this study, we have established a human cancer KB cell line (Kst-6) which stably expressed the CAT gene (pMDRCAT1) driven by the human MDR1 promoter. Exposure to inhibitors of DNA topoisomerase I (camptothecin: CPT-11) and II (etoposide: VP-16 and teniposide: VM-26) could efficiently induce CAT activities in both time- and dose-dependent manners. However, CAT activity could not be significantly induced when treated with an ATP-antagoist, novobiocin. Northern blot analysis showed about 5-fold increase in CAT mRNA levels in Kst-6 cells treated with CPT-11 or VP-16, but not with novobiocin. Proximal MDR1 promoter-binding activities of transacting factor were augmented in nuclear extracts from KB cells treated with CPT-11, VM-26, and VP-16.

Interleukin-4 inhibits lak and initial phase til activity via interleukin-2 receptor.

In the present study, we analyzed the proliferation and cytotoxic activities of LAK cells and initial phase TILs by stimulation with IL-4. IL-4 obviously inhibited the DNA synthesis of LAK cells and initial phase TILs at the concentration of 250 pg/ml and 25 pg/ml, respectively. Furthermore, IL-4 (25 ng/ml for LAK cells, 25 pg/ml for initial phase TILs) suppressed the cytotoxic activities against K562, KATO-III, and autologous tumor cells. The discrepancy of the concentration between the proliferation and the cytotoxicicity by IL-4 suggested different pathways in terms of the generation of LAK cells. In order to clarify the inhibitory mechanism of IL-4, we measured the expression of IL-2 receptor. IL-2 receptor alpha chain was strongly down-regulated by IL-4. Thus, IL-4 modulates the activation of LAK cells and initial phase TILs via the IL-2 receptor alpha chain.

Helicobacter bilis infection in biliary tract cancer.

BACKGROUND: Biliary tract cancer is a highly fatal disease with poor prognosis, but the aetiology is poorly understood. AIM: We aimed to identify Helicobacter bilis infection in the gallbladder in patients with biliary tract disease. METHODS: Archival gallbladder specimens from 34 patients (14 males and 20 females) with an average age of 61.4 +/- 12.2 years (mean +/- SE) were retrieved, consisting of 11 cases of gallbladder cancer, three of bile duct cancer, 16 of cholecystolithiasis and four of pancreatic cancer. DNA was extracted and nested PCR using primers specific for 16S rRNA of H. bilis was performed. RESULTS: Amplification was observed in 3 of 11 gallbladder cancer cases (27.2%) and one of three cases with biliary duct cancer (33.3%). In total, four of 14 cases with biliary tract cancer were positive for H. bilis (28.6%). In addition, the presence of H. bilis was shown in two of 16 cases (12.5%) with cholecystolithiasis. Notably, although the number of cases examined was small, none of the four cases with pancreatic cancer showed the presence of H. bilis infection in the gallbladder without apparent abnormalities. CONCLUSION: H. bilis infection may play a role in biliary tract disease, particularly in biliary tract cancer.

Enhancement of tumor cell susceptibility to tumor-infiltrating lymphocytes by cisplatin.

Some means of enhancing the susceptibility of tumor cells to tumor-infiltrating lymphocytes (TIL) are required in adoptive immunotherapy. This study was designed to investigate whether or not tumor cell lysis by TIL was enhanced by treatment of the tumor cells with cisplatin, and also to clarify the mechanism of cisplatin's action on tumor cells. Autologous tumor cells and established cancer cell lines, including KATO-III and MKN-28, were used. Cytotoxic activities of TIL, the surface antigens of tumor cells, conjugation of TIL and tumor cells, and the production of TNF alpha from TIL were analyzed. Tumor cells treated with 2 micrograms/ml cisplatin for 12 h in vitro were more susceptible to bulk-cultured TIL and TIL clones. The surface antigens of tumor cells were not altered by the treatment with cisplatin. Cisplatin-treated tumor cells showed a higher binding ratio to TIL than did non-treated tumor cells. The anti-(tumor necrosis factor) (anti-TNF) or anti-TNF receptor antibody blocked the enhancement of cytotoxic activity by cisplatin. Thus, it was clarified that cisplatin enhanced the susceptibility of tumor cells to bulk-cultured TIL and TIL clones. Furthermore, the enhancement of cytotoxic activity by TIL in cisplatin-treated tumor cells was caused by a higher binding ratio to TIL and higher susceptibility to the TNF produced by TIL.

Clinical features and management of hepatic portal venous gas: four case reports and cumulative review of the literature.

HYPOTHESIS: Hepatic portal venous gas (HPVG) has been considered a rare entity associated with a grave prognosis. Since 1978, when Liebman et al reviewed 64 cases of HPVG and reported a mortality of 75%, the number of reported cases has been increasing. DESIGN: Case series. PATIENTS AND METHODS: We reviewed the literature on 182 cases of HPVG in adults, including 4 of our patients, (transplantation and abdominal trauma cases were excluded) and analyzed the cause, pathogenesis, and clinical features. RESULTS: In this series, the underlying clinical events associated with HPVG were bowel necrosis (43%), digestive tract dilatation (12%), intraperitoneal abscess (11%), ulcerative colitis (4%), gastric ulcer (4%), Crohn disease (4%), complications of endoscopic procedures (4%), intraperitoneal tumor (3%), and other (15%). The overall mortality was 39% but varied depending on the underlying disease. CONCLUSIONS: Hepatic portal venous gas is a lethal or curable entity caused by various diseases. The underlying disease associated with HPVG determines the clinical features and prognosis of the patients. The treatment of patients with HPVG should be directed to the underlying disease.