Toshiyuki Kitano

Pretreatment neutrophil count as an independent prognostic factor in advanced non-small-cell lung cancer: An analysis of Japan Multinational Trial Organisation LC00-03

We examined the impact of pretreatment neutrophil count on survival in patients with advanced non-small-cell lung cancer (NSCLC). A total of 388 chemo-naïve patients with stage IIIB or IV NSCLC from a randomised controlled trial were evaluated. The effects of pretreatment peripheral blood neutrophil, lymphocyte and monocyte counts and neutrophil-lymphocyte ratio on survival were examined using the proportional hazards regression model to estimate hazard ratios after adjustment for covariates. The optimal cut-off value was determined by proportional hazards regression analysis with the minimum P-value approach and shrinkage procedure. After adjustment for prognostic factors, the pretreatment elevated neutrophil count was statistically significantly associated with short overall (P=0.0008) and progression-free survival (P=0.024), whereas no association was found between prognosis and lymphocyte or monocyte count. The cut-off value selected for neutrophil count was 4500 mm(-3) (corrected hazard ratio, 1.67; 95% confidence interval (CI), 1.09-2.54). The median survival time was 19.3 months (95%CI, 16.5-21.4) for the low-neutrophil group (4500 mm(-3), n=204) and was 10.2 months (95%CI, 8.0-12.3) for the high-neutrophil group (4500 mm(-3), n=184). We confirmed that pretreatment elevated neutrophil count is an independent prognostic factor in patients with advanced NSCLC receiving modern chemotherapy. Neutrophil count is easily measured at low cost, and it may be a useful indicator of patient prognosis.

Astroglial expression of ceramide in Alzheimer's disease brains: a role during neuronal apoptosis.

Accumulating evidences indicate that ceramide is closely involved in apoptotic cell death in neurodegenerative disorders and aging. We examined ceramide levels in the cerebrospinal fluid (CSF) or brain tissues from patients with neurodegenerative disorders and the mechanism of how intra- and extracellular ceramide was regulated during neuronal apoptosis. We screened the ceramide levels in the CSF of patients with neurodegenerative disorders, and found that ceramide was significantly increased in patients with Alzheimers disease (AD) than in patients with age-matched amyotrophic lateral sclerosis (ALS) and other neurological controls. With immunohistochemistry in AD brains, ceramide was aberrantly expressed in astroglia in the frontal cortices, but not detected in ALS and control brains. To explore for the regulation of ceramide in astroglia in Alzheimers disease brains, we examined the metabolism of ceramide during neuronal apoptosis. In retinoic acid (RA)-induced neuronal apoptosis, RA slightly increased de novo synthesis of ceramide, but interestingly, RA dramatically inhibited conversion of [14C] ceramide to glucosylceramide (GlcCer), suggesting that the increase of ceramide mass is mainly due to inhibition of the ceramide-metabolizing enzyme GlcCer synthase. In addition, a significant increase of the [14C] ceramide level in the culture medium was detected by chasing and turnover experiments without alteration of extracellular [14C] sphingomyelin levels. A 2.5-fold increase of ceramide mass in the supernatant was also detected after 48 h of treatment with RA. These results suggest a regulatory mechanism of intracellular ceramide through inhibition of GlcCer synthase and a possible role of ceramide as an extracellular/intercellular mediator for neuronal apoptosis. The increased ceramide level in the CSF from AD patients, which may be derived from astroglia, raises a possibility of neuronal apoptosis by the response to intercellular ceramide in AD.

Diversity and complexity of ceramide signalling in apoptosis.

Sphingolipid ceramide has emerged as a lipid messenger of cell functions including differentiation and apoptosis. Diverse kinds of stresses (ultraviolet, irradiation, heat shock and hypoxia) and biological factors (TNF-alpha, IFN-gamma and Fas antibody) require ceramide generation to execute apoptosis. The review summarises the diversity and complexity of up- and downstream of ceramide signalling in apoptosis and clinical implications of ceramide-induced apoptosis.

Increase of Nuclear Ceramide through Caspase-3-Dependent Regulation of the “Sphingomyelin Cycle” in Fas-Induced Apoptosis

Regardless of the existence of ceramide-related molecules, such as sphingomyelin (SM), neutral sphingomyelinase (nSMase), and SM synthase, in the nucleus, the regulation of ceramide in the nucleus is poorly understood in stress-induced apoptosis. In Fas-induced Jurkat T-cell apoptosis, we found a time- and dose-dependent increase of ceramide content in the nuclear and microsomal fractions. Fas-induced increase of ceramide content in the nucleus also was detected by confocal microscopy using anticeramide antibody. Activation of nSMase and inhibition of SM synthase were evident in the nuclear fraction after Fas cross-linking, whereas nSMase was activated, but SM synthase was not affected, in the microsomal fraction. Pretreatment with d-609, a putative SM synthase inhibitor, enhanced Fas-induced increase of ceramide in the nucleus and induction of apoptosis along with increase of Fas-induced inhibition of nuclear SM synthase. Fas-induced activation of caspase-3 was detected in the nuclear fraction and in whole cell lysate. A caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-chloromethyl ketone, blocked not only Fas-induced increases of apoptosis and ceramide content but also Fas-induced activation of nSMase and inhibition of SM synthase in the nuclear fraction. Taken together, it is suggested that the nucleus is a site for ceramide increase and caspase-3 activation in Fas-induced Jurkat T-cell apoptosis and that caspase-3-dependent regulation of the “SM cycle” consisting of nSMase and SM synthase plays a role in Fas-induced ceramide increase in the nucleus.

Serum Neutrophil Extracellular Trap Levels Predict Thrombotic Microangiopathy after Allogeneic Stem Cell Transplantation

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a devastating complication of hematopoietic stem cell transplantation. TA-TMA likely represents the final stage of vascular endothelial injury; however, its pathophysiology is largely unknown, making clinical management difficult. Recently, the association of neutrophil extracellular traps (NETs) with the development of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome has been reported. Thus, we explored whether NETs are also relevant to the occurrence of TA-TMA. We retrospectively analyzed post-transplant trends of serum NET levels in 90 patients, 11 of whom developed TA-TMA. Relative to baseline (before the conditioning regimen), elevated serum NET levels either at 4 weeks after transplantation or as early as the day of transplantation were associated with significantly increased risk of TA-TMA. In contrast, thrombomodulin, a potential marker for TA-TMA, was not helpful to predict the occurrence of TA-TMA in our study. In addition, we directly detected glomerular deposition of NETs in 2 TA-TMA patients. Increased NET levels are a significant risk factor for TA-TMA, suggesting that NET level is a useful biomarker for TA-TMA.

A Clinical, Pathological, and Genetic Characterization of Methotrexate-associated Lymphoproliferative Disorders

Objective. Methotrexate-associated lymphoproliferative disorders (MTX-LPD) often regress spontaneously during MTX withdrawal, but the prognostic factors remain unclear. The aim of our study was to clarify the clinical, histological, and genetic factors that predict outcomes in patients with MTX-LPD. Methods. Patients with MTX-LPD diagnosed between 2000 and 2012 were analyzed retrospectively regarding their clinical course, site of biopsy, histological typing, Epstein-Barr virus (EBV) in situ hybridization and immunostaining, and HLA type. Results. Twenty-one patients, including 20 with rheumatoid arthritis (RA) and 1 with polymyositis, were analyzed. The mean dose of MTX was 6.1 mg/week and the mean duration of treatment was 71.1 months. Clinically, 5 patients were diagnosed with EBV-positive mucocutaneous ulcer (EBVMCU) and had polymorphic histological findings. The proportion of those patients successfully treated solely by withdrawal of MTX was significantly greater than that of those without EBVMCU (75% vs 7.7%, p = 0.015). The HLA-B15:11 haplotype was more frequent in patients with EBV+ RA with MTX-LPD than in healthy Japanese controls (p = 0.0079, Bonferroni’s method). EBV latency classification and HLA typing were not associated with the prognosis of MTX-LPD in our cohort. Conclusion. Our data demonstrate that patients in the EBVMCU, a specific clinical subgroup of MTX-LPD, had a better clinical outcome when MTX was withdrawn than did other patients with MTX-LPD.

Control of ceramide-induced apoptosis by IGF-1: involvement of PI-3 kinase, caspase-3 and catalase

Insulin-like growth factor-1 (IGF-1) inhibited N-acetylsphingosine (C2-ceramide)-induced HL-60 cell apoptosis via relieving oxidative damage. This inhibitory action of IGF-1 was blocked by a phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin and enhanced by overexpression of the p110 catalytic subunit of PI-3 kinase. Either IGF-1 pretreatment or PI-3 kinase overexpression restored ceramide-depleted catalase function, and this restoration was inhibited by wortmannin. A catalase inhibitor 3-amino-1h-1, 2, 4-triazole (ATZ) blocked the inhibitory action of IGF-1 on ceramide-induced apoptosis, whereas exogenous purified catalase enhanced it. Ceramide-activated caspase-3 was inhibited by IGF-1/PI-3 kinase and enhanced by wortmannin, while the addition of a specific caspase-3 inhibitor DMQD-CHO significantly enhanced the restoration by IGF-1 of ceramide-depleted catalase function. Moreover, IGF-1 inhibited C2-ceramide-induced decrease of mitochondrial membrane potential, and increase of cytochrome c release, caspase-3 cleavage and caspase-3 activity as judged by PhiPhiLux cleaving method. In summary, these results suggest that IGF-1/PI-3 kinase inhibited C2-ceramide-induced apoptosis due to relieving oxidative damage, which resulted from the inhibition of catalase by activated caspase-3.

Associations between glutathione S-transferase π Ile105Val and glyoxylate aminotransferase Pro11Leu and Ile340Met polymorphisms and early-onset oxaliplatin-induced neuropathy

PURPOSE Although the risk of oxaliplatin-induced neuropathy depends on cumulative oxaliplatin dose, susceptibility to this adverse event differs greatly among patients. In this study, we investigated the associations between oxaliplatin-induced neuropathy and the following polymorphisms: glutathione S-transferase pi (GSTP1) Ile(105)Val, and glyoxylate aminotransferase (AGXT) Pro(11)Leu and AGXT Ile(340)Met. EXPERIMENTAL DESIGN Eighty-two Japanese patients with histologically confirmed colorectal cancer who received at least six cycles of the modified FOLFOX6 (m-FOLFOX6) regimen were enrolled. To minimize differences in cumulative oxaliplatin dose between patients, oxaliplatin-induced neuropathy was evaluated using an oxaliplatin-specific scale during the 2-week period after completion of the sixth cycle of treatment. RESULTS Forty-four patients developed grade 2/3 oxaliplatin-induced neuropathy. There were more patients carrying at least one GSTP1(105)Val allele among the group with grade 2/3 neuropathy (18/44, 41%) than among the group with grade 1 neuropathy (9/38, 24%), although the difference was not statistically significant (P=0.098). There were similar numbers of patients carrying at least one AGXT(105)Met allele in the grade 2/3 neuropathy (7/44, 16%) and grade 1 neuropathy groups (5/38, 13%; P=0.725). The AGXT(11)Leu allele was not found in any of our patients or controls. CONCLUSIONS We found no significant association between oxaliplatin-induced neuropathy and the GSTP1 Ile(105)Val and AGXT Ile(340)Met polymorphisms. Given that no AGXT(11)Leu allele was found among our study population (n=177), evaluating this polymorphism in Japanese patients in future studies is likely to be uninformative.

Single-Agent Gemcitabine for Biliary Tract Cancers

Objective: The aim of this study was to investigate the outcomes of gemcitabine-treated patients with inoperable biliary tract cancers. Methods: We conducted a retrospective study of consecutively treated 22 inoperable biliary tract cancer patients with gemcitabine (500–1,000 mg/m2 on days 1, 8, 15 every 4 weeks) as first-line, and 17 patients as second- or third-line treatment. Results: The response rate of patients treated with gemcitabine as first-line and second- or third-line treatment was 5.3 and 28.5%, respectively. The median overall survival time in the first-, and second- or third-line treatment groups was 8.3 and 17.0 months, and the 1-year survival rate was 44.0 and 50.9%, respectively. The present study also suggests the possibility that the prognosis of patients with high levels of C-reactive protein and total bilirubin, or a low level of albumin might be worse. Conclusions: Our results indicate that the treatment of inoperable biliary tract cancers with gemcitabine is feasible. There was no difference in the response rate and overall survival between biliary tract cancer patients in the first- and second- or third-line treatment groups. We also present the systematic review of literature of the recent treatment results of biliary tract cancers treated with gemcitabine.

Oxaliplatin-Free Interval as a Risk Factor for Hypersensitivity Reaction among Colorectal Cancer Patients Treated with FOLFOX

Background: Hypersensitivity reaction (HSR) and sensory neuropathy are major complications of oxaliplatin-based chemotherapy. Preplanned withdrawal of oxaliplatin after the first six cycles and reintroduction at the time of disease progression (stop-and-go strategy) may reduce neurotoxicity. However, the effect of an oxaliplatin-free interval on HSR occurrence remains poorly understood. Patients and Methods: Data on patients with colorectal cancer who received FOLFOX (5-fluorouracil, leucovorin and oxaliplatin) treatment between June 2005 and June 2009 were retrieved from the prospective cohort database of the Outpatient Oncology Unit of the Kyoto University Hospital. Factor analysis was performed. Results: Among patients who received six or fewer cycles of FOLFOX, the incidence of HSR was low (7/99, 7.1%). For patients who received more than six cycles, the incidence of HSR was higher among patients treated with stop-and-go FOLFOX than among patients treated with continuous FOLFOX (25/61, 41.0% vs. 13/63, 20.6%; p = 0.019). Interestingly, most cases of HSR during stop-and-go FOLFOX occurred during the second or third cycle of the reintroduction phase (21/25, 84%). Multivariate analysis identified undergoing an oxaliplatin-free interval as an independent risk factor (p = 0.016). Conclusions: An oxaliplatin-free interval may increase the risk of HSR. Special vigilance is needed during the second and third cycles after reintroduction of oxaliplatin.