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

BACKGROUND: We aimed to compare the efficacy and safety of irinotecan/S-1 (IRIS) therapy with S-1 monotherapy in patients with gemcitabine-refractory pancreatic cancer. METHODS: Patients were treated with oral S-1 (80-120 mg for 14 days every 4 weeks) plus intravenous irinotecan (100 mg m-2 on days 1 and 15 every 4 weeks; IRIS group) or oral S-1 group (80-120 mg daily for 28 days every 6 weeks). The primary endpoint was progression-free survival (PFS). RESULTS: Of 137 patients enrolled, 127 were eligible for efficacy. The median PFS in the IRIS group and S-1 monotherapy group were 3.5 and 1.9 months, respectively (hazard ratio (HR)=0.77; 95% confidence interval (CI), 0.53-1.11; P=0.18), while the median overall survival (OS) were 6.8 and 5.8 months, respectively (HR=0.75; 95% CI, 0.51-1.09; P=0.13). Response rate was significantly higher in the IRIS group than in the S-1 monotherapy group (18.3% vs 6.0%, P=0.03). Grade 3 or higher neutropenia and anorexia occurred more frequently in the IRIS group. CONCLUSIONS: There was a trend for better PFS and OS in the IRIS group that could be a treatment arm in the clinical trials for gemcitabine-refractory pancreatic cancer.

New approaches to gene and cell therapy for hemophilia.

Hemophilia is considered suitable for gene therapy because it is caused by a single gene abnormality, and therapeutic coagulation factor levels may vary across a broad range. Recent success of hemophilia B gene therapy with an adeno-associated virus (AAV) vector in a clinical trial showed the real prospect that, through gene therapy, a cure for hemophilia may become a reality. However, AAV-mediated gene therapy is not applicable to patients with hemophilia A at present, and neutralizing antibodies against AAV reduce the efficacy of AAV-mediated strategies. Because patients that benefit from AAV treatment (hemophilia B without neutralizing antibodies) are estimated to represent only 15% of total patients with hemophilia, the development of basic technologies for hemophilia A and those that result in higher therapeutic effects are critical. In this review, we present an outline of gene therapy methods for hemophilia, including the transition of technical developments thus far and our novel techniques.

An internally and externally validated nomogram for predicting the risk of irinotecan-induced severe neutropenia in advanced colorectal cancer patients.

BACKGROUND: In Asians, the risk of irinotecan-induced severe toxicities is related in part to UGT1A1*6 (UGT, UDP glucuronosyltransferase) and UGT1A1*28, variant alleles that reduce the elimination of SN-38, the active metabolite of irinotecan. We prospectively studied the relation between the UGT1A1 genotype and the safety of irinotecan-based regimens in Japanese patients with advanced colorectal cancer, and then constructed a nomogram for predicting the risk of severe neutropenia in the first treatment cycle. METHODS: Safety data were obtained from 1312 patients monitored during the first 3 cycles of irinotecan-based regimen in a prospective observational study. In development of the nomogram, multivariable logistic regression analysis was used to test the associations of candidate factors to severe neutropenia in the first cycle. The final nomogram based on the results of multivariable analysis was constructed and validated internally using a bootstrapping technique and externally in an independent data set (n=350). RESULTS: The UGT1A1 genotype was confirmed to be associated with increased risks of irinotecan-induced grade 3 or 4 neutropenia and diarrhoea. The final nomogram included type of regimen, administered dose of irinotecan, gender, age, UGT1A1 genotype, Eastern Cooperative Oncology Group performance status, pre-treatment absolute neutrophil count, and total bilirubin level. The model was validated both internally (bootstrap-adjusted concordance index, 0.69) and externally (concordance index, 0.70). CONCLUSIONS: Our nomogram can be used before treatment to accurately predict the probability of irinotecan-induced severe neutropenia in the first cycle of therapy. Additional studies should evaluate the effect of nomogram-guided dosing on efficacy in patients receiving irinotecan.

Production of functional coagulation factor VIII from iPSCs using a lentiviral vector.

The use of induced pluripotent stem cells (iPSCs) as an autologous cell source has shed new light on cell replacement therapy with respect to the treatment of numerous hereditary disorders. We focused on the use of iPSCs for cell-based therapy of haemophilia. We generated iPSCs from mesenchymal stem cells that had been isolated from C57BL/6 mice. The mouse iPSCs were generated through the induction of four transcription factor genes Oct3/4, Klf-4, Sox-2 and c-Myc. The derived iPSCs released functional coagulation factor VIII (FVIII) following transduction with a simian immunodeficiency virus vector. The subcutaneous transplantation of iPSCs expressing FVIII into nude mice resulted in teratoma formation, and significantly increased plasma levels of FVIII. The plasma concentration of FVIII was at levels appropriate for human therapy at 2-4 weeks post transplantation. Our data suggest that iPSCs could be an attractive and prospective autologous cell source for the production of coagulation factor, and that engineered iPSCs expressing coagulation factor might provide a cell-based therapeutic strategy appropriate for haemophilia.

A case of everolimus-associated chylothorax in a cardiac transplant recipient.

We herein report a case of putative everolimus-associated chylothorax in a cardiac transplant recipient. A 17-year-old Japanese boy with dilated cardiomyopathy and severe cardiac failure requiring left ventricular assist support was determined to be a cardiac transplant candidate in 1992. He underwent overseas heart transplantation in Houston, Texas in October 1992. He was subsequently treated with immunosuppression therapy: Cyclosporine (CSA), azathioprine, and prednisolone (PRD). After several acute rejection episodes requiring steroid therapy, intravascular ultrasonography revealed a moderate degree of transplant coronary arterial vasculopathy (TCAV) with 50% stenosis in 2003. He underwent coronary stenting twice; the immunosuppressive regimen was converted to CSA, mycophenolate mofetil, everolimus (EVL), and PRD in 2006. TCAV has not progressed since then. In October 2008, chest x-ray showed bilateral pleural effusion. As we thought that the pleural effusion was caused by cardiac dysfunction due to moderate mitral regurgitation and TCAV as well as renal impairment, he was treated with diuretics and digoxin. However, the pleural effusion progressed gradually associated with exertional dyspnea and moderate edema of his lower legs. Chest computed tomography showed massive bilateral pleural effusions without evidence of malignancy in 2011. A pleural tap in 2011 revealed chylothorax. Although mammalian target of rapamycin inhibitors were major drugs for lymphoangioleimyomatosis, we believed that the chylothorax was associated with EVL. EVL was discontinued in March 2011: the chylothorax spontaneously resolved in November 2011.

Intra-articular injection of mesenchymal stem cells expressing coagulation factor ameliorates hemophilic arthropathy in factor VIII-deficient mice.

BACKGROUND: Transplantation of cells overexpressing a target protein represents a viable gene therapeutic approach for treating hemophilia. Here, we focused on the use of autologous mesenchymal stem cells (MSCs) expressing coagulation factor for the treatment of coagulation factor VIII (FVIII) deficiency in mice. METHODS AND RESULTS: Analysis of luciferase gene constructs driven by different promoters revealed that the plasminogen activator inhibitor-1 (PAI-1) gene promoter coupled with the cytomegalovirus promoter enhancer region was one of the most effective promoters for producing the target protein. MSCs transduced with the simian immunodeficiency virus (SIV) vector containing the FVIII gene driven by the PAI-1 promoter expressed FVIII for several months, and this expression was maintained after multiple mesenchymal lineage differentiation. Although intravenous injection of cell supernatant derived from MSCs transduced with an SIV vector containing the FVIII gene driven by the PAI-1 promoter significantly increased plasma FVIII levels, subcutaneous implantation of the MSCs resulted in a transient and weak increase in plasma FVIII levels in FVIII-deficient mice. Interestingly, intra-articular injection of the transduced MSCs significantly ameliorated the hemarthrosis and hemophilic arthropathy induced by knee joint needle puncture in FVIII-deficient mice. The therapeutic effects of a single intra-articular injection of transduced MSCs to inhibit joint bleeding persisted for at least 8 weeks after administration. CONCLUSIONS: MSCs provide a promising autologous cell source for the production of coagulation factor. Intra-articular injection of MSCs expressing coagulation factor may offer an attractive treatment approach for hemophilic arthropathy.

Lack of IL-21 signal attenuates graft-versus-leukemia effect in the absence of CD8 T-cells.

Previously, we have shown that IL-21R(-/-) splenocytes ameliorate GVHD as compared with wild-type splenocytes. Here, we investigated whether or not IL-21R(-/-) splenocytes diminish the graft-versus-leukemia (GVL) effect. Surprisingly, IL-21R(-/-) splenocytes efficiently eliminate leukemic cells as well as wild-type splenocytes, suggesting the retention of GVL effects in the absence of IL-21 signaling. To compare the GVL effect between IL-21R(-/-) and wild-type cells, we titrated the number of splenocytes required for the elimination of leukemic cells and found that the threshold of GVL effect was obtained between 5 × 10(5) and 5 × 10(6) with both types of splenocytes. Cotransplantation with CD8-depleted splenocytes but not with purified CD8 T-cells resulted in a significant reduction in anti-leukemic effect of IL-21R(-/-) cells compared with wild-type cells, suggesting that the lack of IL-21 signaling primarily impairs CD4 T-cell rather than CD8 T-cell function and the comparable GVL effect with IL-21R(-/-) bulk splenocytes results from cooperative compensation by CD8 T-cells.

Hepatic arterial infusion of mitomycin C with degradable starch microspheres for unresectable intrahepatic cholangiocarcinoma.

AIMS: With no effective chemotherapy, the prognosis of unresectable intrahepatic cholangiocarcinoma is extremely poor. Hepatic arterial infusion of mitomycin C with degradable starch microspheres has been reported to be an effective treatment for unresectable liver metastasis. We retrospectively evaluated the efficacy and safety of this chemotherapy for treating unresectable intrahepatic cholangiocarcinoma. MATERIALS AND METHODS: Hepatic arterial infusion chemotherapy through an implanted port system was carried out in 20 patients with unresectable intrahepatic cholangiocarcinoma. Degradable starch microspheres mixed with mitomycin C and contrast media were injected until either embolisation of the hepatic artery or influx to the gastroduodenal system was confirmed. This treatment was repeated weekly. RESULTS: Hepatic arterial infusion chemotherapy was carried out 204 times. The response rate was 50.0%. Twelve patients experienced transient epigastralgia and four experienced gastroduodenal ulcer. The time to progression was 8.3 months and the median survival time was 14.1 months. CONCLUSIONS: This chemotherapy was effective and feasible for patients with unresectable intrahepatic cholangiocarcinoma. Further study with a larger number of patients is warranted.

Phase I/II study of docetaxel and S-1 in patients with advanced gastric cancer.

The aims of this phase I/II study of docetaxel and S-1 were to determine the dose-limiting toxicity (DLT), maximum-tolerated dose (MTD), and recommended dose (RD) in the phase I part and to explore the tumour response, survival and safety in the phase II part. Patients with histologically- or cytologically confirmed unresectable or recurrent gastric cancer were eligible. Treatment consisted of intravenous docetaxel on day 1 (starting dose 50 mg m(-2)) and oral S-1 at a fixed dose of 40 mg m(-2) twice daily on days 1-14, every 4 weeks up to six cycles. Nine patients took part in the phase I portion of the study. The MTD of docetaxel was determined to be 50 mg m(-2), with the DLTs of grade 3 infection associated with grade 3 neutropenia and grade 4 neutropenia during S-1 administration. The RD of docetaxel was 40 mg m(-2) in combination with S-1 40 mg m(-2) b.i.d. The efficacy and safety of this regimen was therefore assessed in 46 patients with at least one measurable lesion. The overall response rate and estimated median overall survival were 46% (95% CI, 31-61%) and 14.0 months (8.3-17.3 months), respectively. The most common grade 3/4 toxicity was neutropenia (67% of patients), which was predictable and manageable. This regimen showed promising activity with moderate toxicities in advanced gastric cancer.

Aspirin resistance detected with aggregometry cannot be explained by cyclooxygenase activity: involvement of other signaling pathway(s) in cardiovascular events of aspirin-treated patients.

OBJECTIVES: Although the concept of aspirin resistance is extensively reported in medical literature, its precise mechanisms and clinical outcomes are largely unknown. In this study, we examined individual thromboxane biosynthesis and platelet aggregation in aspirin-treated patients, and whether the results of a platelet aggregation test influenced clinical outcomes. RESULTS: Subjects taking 81 mg of aspirin (n = 50) and controls (n = 38) were evaluated for platelet aggregation and platelet cyclooxygenase-1 (COX-1) activity by measuring collagen-induced thromboxane B2 production. For aggregometry, both light transmission (LT) and laser-light scattering methods were employed to quantitatively evaluate aggregate sizes and numbers. Aspirin treatment resulted in the inhibition of collagen-induced platelet aggregation, particularly the transition from small to large platelet aggregates. Although platelet COX-1 activity seemed to be uniformly inhibited in all patients, platelet aggregation studies showed great inter-individual differences; variation in platelet COX-1 activity only accounted for 6-20% of the individual aggregations. Factor analysis revealed the existence of a common factor (other than platelet COX-1) that explained 48.4% of the variations in platelet aggregation induced by collagen, adenosine diphosphate (ADP), and collagen-related peptide. We then prospectively enrolled 136 aspirin-treated patients in our study, and we found that being in the upper quartile level of LT, or with large aggregate formation induced by collagen, was an independent risk factor for developing cardiovascular events within 12 months [hazard ratio (HR) = 7.98, P = 0.008 for LT; HR = 7.76, P = 0.007 for large aggregates]. On the other hand, the existence of diabetes mellitus was an independent risk factor for overall outcomes (HR 1.30-11.9, P = 0.015-0.033). CONCLUSIONS: Aspirin resistance expressed as unsuppressed platelet COX-1 activity is a rare condition in an out-patient population. Other factor(s) affecting collagen-induced platelet aggregation may influence early outcomes in aspirin-treated patients.

The squamous cell carcinoma antigens as relevant biomarkers of atopic dermatitis.

BACKGROUND: Although it is thought that both Th1- and Th2-type inflammations are involved in the pathogenesis of atopic dermatitis (AD), it is controversial which immune response is more involved in regulating the clinical severity of AD. We recently found that the squamous cell carcinoma antigens 1 (SCCA1) and SCCA2 are novel biomarkers of bronchial asthma, downstream of IL-4 and IL-13. OBJECTIVE: We examined whether SCCA1 and SCCA2 could also serve as biomarkers of AD, reflecting its Th2-type immune responses, and whether the expression level of SCCA was correlated with clinical severity of AD. METHOD: We compared the expression of SCCA1 and SCCA2 at the mRNA and protein levels in both involved and uninvolved skin of AD patients and in normal control skin. We next analysed induction of SCCA by IL-4 or IL-13 in keratinocytes. Finally, we compared the serum level of SCCA with laboratory parameters reflecting Th2-type inflammation and clinical severity in AD patients. RESULTS: SCCA1 and SCCA2 were highly expressed in involved skin of AD patients, compared with their uninvolved skin, at both mRNA and protein levels. SCCA protein was dominantly expressed in suprabasal keratinocytes in the epidermis of AD patients. Either IL-4 or IL-13, but not IFN-gamma or TNF, induced production of SCCA in keratinocytes. These result suggest that SCCA is induced in AD skin, probably due to direct actions of IL-4 and/or IL-13 on keratinocytes. Serum levels of SCCA were well correlated with eosinophil numbers and serum lactate dehydrogenase levels, and weakly with serum IgE levels, in AD patients. Furthermore, serum levels of SCCA were strongly correlated with clinical severity. CONCLUSIONS: Th2-type inflammation dominantly regulates the clinical severity of AD, and SCCA is a relevant biomarker of AD, reflecting both Th2-type inflammation and clinical severity.

Characterization of novel squamous cell carcinoma antigen-related molecules in mice.

The squamous cell carcinoma antigen 1 (SCCA1) and SCCA2 are unique serpins that can inhibit cysteine proteinases. SQN-5, their mouse ortholog, has already been identified, and its inhibitory property has been characterized; however, its biological role has remained undefined. Furthermore, no other mouse homolog of SQN-5 has been known. We characterize three mouse members of SCCA-related molecules including SQN-5 in this article. Serpinb3a (SQN-5) and Serpinb3b, but not Serpinb3c, were functional, inhibiting both serine and cysteine proteinases with different inhibitory profiles due to the difference of two amino acids in their reactive site loops. Serpinb3a was ubiquitously expressed in most tissues, whereas expression of Serpinb3b was limited to keratinocytes. Keratinocytes secreted both SCCA-related proteins, Serpinb3a and Serpinb3b. These results indicate that Serpinb3a and Serpinb3b may play different roles by inhibiting intrinsic or extrinsic proteinases with different expression distributions and different inhibitory profiles.

Materno-fetal coordination of stress-induced Fos expression in the hypothalamic paraventricular nucleus during pregnancy.

This study investigates whether maternal stress during pregnancy induces maternal and fetal hypothalamic paraventricular nucleus (PVN) neuronal activation and the effects of maternal stress on fetal hypothalamic and PVN brain-derived neurotrophic factor (BDNF) expression. Pregnant rats were exposed to three types of maternal stress with varying severity (restraint, forced walking and immobilization) for 30 min on gestational day 21. Severity of stress was assessed by measurement of maternal plasma corticosterone 30 min following the stimulus. Maternal plasma corticosterone increased in each stress response group (immobilization>forced walking>restraint). Further, the expression of Fos protein, a marker of neuronal activation, increased in the fetal and maternal PVN in direct relation to the severity of stress treatments. Forced walking and immobilized stress, but not restraint stress, significantly increased BDNF expression in the fetal hypothalamus.These findings suggest that the fetal hypothalamic-pituitary-adrenal (HPA) response following maternal stress mirrors maternal HPA activation. In addition, BDNF may play a role in protecting fetal brain neurons from damage caused by severe stress.

Sesquiterpenoids in root exudates of Solanum aethiopicum.

Five known sesquiterpenoids, solavetivone, lubimin, lubiminoic acid, aethione and lubiminol were isolated from the root exudates recovered from Solanum aethiopicum by a newly proposed method using charcoal. Quantitative analysis of the sesquiterpenoids in the root exudates of S. aethiopicum and S. melongena suggested that relatively large amounts of the sesquiterpenoids were exuded from the roots. Antifungal activity of the sesquiterpenoids against Fusarium oxysporum and Verticillium dahliae was also examined.

Sequence analysis of fibroblast growth factor receptor 2 ( FGFR2 ) in Japanese patients with craniosynostosis.

Recently, mutations of the fibroblast growth factor receptor ( FGFR ) genes have been detected in syndromic craniosynostosis. We examined nucleotide sequences of FGFR2 in Japanese craniosynostosis patients (Crouzon syndrome: 9 cases; Apert syndrome: 6 cases; scaphocephaly: 3 cases as non-syndromic patients) by polymerase chain reaction (PCR) followed by direct sequencing methods. The results demonstrated FGFR2 heterozygous mutations at codons 252, 290 of exon 7, and at codon 342, 354 of exon 9 in Crouzon syndromes. In Apert syndrome patients, Ser252Trp and Pro253Arg were detected in five and one patients, respectively. No mutation was detected in one case of Crouzon, all cases of scaphocephaly and healthy individuals. Thus far sequence analysis of FGFR2 in syndromic craniosynostosis has been reported in many white patients, whereas in Japanese only several cases have been studied. The current study with 18 patients confirmed that a similar series of mutations occur in Japanese patients as in white patients regardless of ethnicity and environment. The frequency of the mutation was 82% (9/11 cases) in Japanese Crouzon patients. The ratio of S252W:P253R was 5 : 1 in Japanese Apert patients. Moreover, in Japanese Apert patients, complication rate of cleft palate was 60% for mutation of Ser252Trp and 0 of 2 patients for Pro253Arg, with their syndactyly score being 4.90 and 5.50, respectively.

[Standard therapy of CPT-11 for colorectal cancer].

Irinotecan (CPT-11) is a potent inhibitor of topoisomerase I, and has demonstrated antitumor activity against metastatic colorectal cancer. In phase II studies, CPT-11 showed promising activity. Combining irinotecan with 5-fluorouracil (5-FU) and Leucovorin (LV) has shown benefits for patients with metastatic colorectal cancer CPT-11/5-FU/LV therapy is considered as a standard therapy. Weekly or biweekly administration of CPT-11 is most recommended.

Complete suppression of insulitis and diabetes in NOD mice lacking interferon regulatory factor-1.

Interferon regulatory factor-1 (IRF-1), a transcriptional factor, regulates type I interferon and interferon-induced genes. It was reported that IRF-1 regulates important molecules required for inflammation and immune reactions. To investigate the role of IRF-1 in the development of autoimmune diabetes, we established IRF-1 deficient (IRF-1(-/-)) non-obese diabetic (NOD) mice. IRF-1-deficient C57BL/6J mice were out-crossed to NOD mice, and F1 were backcrossed to NOD mice. At the N8 generation, the heterozygote for IRF-1 mutation was intercrossed and N8F1 was obtained. Out of three NOD genotypes, IRF-1(+/+) and IRF-1(+/-) developed spontaneous diabetes with an incidence of 47% (9/19) and 50% (10/20) by 30 weeks of age, respectively; whereas IRF-1(-/-) did not develop diabetes (0/18, P< 0.01 vs. (+/+) and (+/-)). Histologically, IRF-1(+/+) and IRF-1(+/-) had various degrees of insulitis, but IRF-1(-/-) had no insulitis. In comparison with IRF-1(+/+), the percentage of CD4(+) and Mac-1(+) splenic cells significantly increased, whereas CD3(+), CD8(+) and B220(+) cells decreased in IRF-1(-/-). Furthermore, spleen cell proliferation in response to Con A or murine GAD65 peptide, a major autoantigen of the pancreatic beta-cell, significantly increased, and the IFN-gamma/IL-10 ratio in the culture supernatant significantly decreased in IRF-1(-/-), suggesting Th2 deviation in cytokine balance. These results indicate that IRF-1 plays a key role in developing insulitis and diabetes in NOD mice.