BioBusiness Group

Tokyo, Japan

BioBusiness Group

Tokyo, Japan
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Murakoshi Y.,National Cancer Center Research Institute | Murakoshi Y.,Tokyo Medical University | Honda K.,National Cancer Center Research Institute | Sasazuki S.,Research Center for Cancer Prevention and Screening | And 18 more authors.
Cancer Science | Year: 2011

The development of a new plasma biomarker for early detection would be necessary to improve the overall outcome of colorectal cancer. Here we report the identification and validation of the ninth component of complement (C9) as a novel plasma biomarker for colorectal cancer by cutting-edge proteomic technologies. Plasma proteins were enzymatically digested into a large array of peptides, and the relative quantity of a total of 94803 peptide peaks was compared between 31 colorectal cancer patients and 59 age/sex-matched healthy controls using 2D image-converted analysis of liquid chromatography and mass spectrometry. The selected biomarker candidates were validated in 345 subjects (115 colorectal cancer patients and 230 age/sex-matched healthy controls) using high-density reverse-phase protein microarrays. Plasma levels of Apo AI and C9 in colorectal cancer patients significantly differed from healthy controls with P values of 7.94×10-4 and 1.43×10-12 (Student's t-test), respectively. In particular, C9 was elevated in patients with colorectal cancer, including those with stage-I and -II diseases (P=3.01×10-3 and P=1.13×10-5, respectively). Although the significance of the present study must be validated in an independent clinical study, the increment of plasma C9 may be applicable to the early detection of colorectal cancer. © 2010 Japanese Cancer Association.


Matsubara J.,National Cancer Center Research Institute | Matsubara J.,Kyoto University | Ono M.,National Cancer Center Research Institute | Honda K.,National Cancer Center Research Institute | And 15 more authors.
Molecular and Cellular Proteomics | Year: 2010

Although gemcitabine monotherapy is the standard treatment for advanced pancreatic cancer, patient outcome varies significantly, and a considerable number do not benefit adequately.We therefore searched for new biomarkers predictive of overall patient survival. Using LC-MS, we compared the base-line plasma proteome between 29 representative patients with advanced pancreatic cancer who died within 100 days and 31 patients who survived for more than 400 days after receiving at least two cycles of the same gemcitabine monotherapy. Identified biomarker candidates were then challenged in a larger cohort of 304 patients treated with the same protocol using reverse-phase protein microarray. Among a total of 45,277 peptide peaks, we identified 637 peaks whose intensities differed significantly between the two groups (p < 0.001, Welch's t test). Two MS peaks with the highest statistical significance (p = 2.6 × 10 -4 and p = 5.0 × 10-4) were revealed to be derived from α1-antitrypsin and α1-antichymotrypsin, respectively. The levels of α1-antitrypsin (p = 8.9 × 10-8) and α1-antichymotrypsin (p = 0.001) were significantly correlated with the overall survival of the 304 patients. We selected α1-antitrypsin (p = 0.0001), leukocyte count (p = 0.066), alkaline phosphatase (p = 8.3 × 10-8), and performance status (p = 0.003) using multivariate Cox regression analysis and constructed a scoring system (nomogram) that was able to identify a group of high risk patients having a short median survival time of 150 days (95% confidence interval, 123-187 days; p = 2.0 × 10 -15, log rank test). The accuracy of this model for prognostication was internally validated and showed good calibration and discrimination with a bootstrap-corrected concordance index of 0.672. In conclusion, an increased level of α1-antitrypsin is a biomarker that predicts short overall survival of patients with advanced pancreatic cancer receiving gemcitabine monotherapy. Although an external validation study will be necessary, the current model may be useful for identifying patients unsuitable for the standardized therapy. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Matsubara J.,National Cancer Center Research Institute | Matsubara J.,Kyoto University | Honda K.,National Cancer Center Research Institute | Ono M.,National Cancer Center Research Institute | And 16 more authors.
Cancer Epidemiology Biomarkers and Prevention | Year: 2011

Background: The aim of this study was to identify a new plasma biomarker for use in early detection of colorectal cancer. Methods: Using the combination of hollow fiber membrane (HFM)-based low-molecular weight protein enrichment and two-dimensional image converted analysis of liquid chromatography and mass spectrometry (2DICAL), we compared the plasma proteome of 22 colorectal cancer patients with those of 21 healthy controls. An identified biomarker candidate was then validated in two larger cohorts [validation-1 (n = 210) and validation-2 (n = 113)] using a high-density reverse-phase protein microarray. Results: From a total of 53,009 mass peaks, we identified 103 with an area under curve (AUC) value of 0.80 or higher that could distinguish cancer patients from healthy controls. A peak that increased in colorectal cancer patients, with an AUC of 0.81 and P value of 0.0004 (Mann-Whitney U test), was identified as a product of the PLIN2 gene [also known as perilipin-2, adipose differentiation-related protein (ADRP), or adipophilin]. An increase in plasma adipophilin was consistently observed in colorectal cancer patients, including those with stage I or stage II disease (P < 0.0001, Welch's t test). Immunohistochemical analysis revealed that adipophilin is expressed primarily in the basal sides of colorectal cancer cells forming polarized tubular structures, and that it is absent from adjacent normal intestinal mucosae. Conclusions: Adipophilin is a plasma biomarker potentially useful for the detection of early-stage colorectal cancer. Impact: The combination of HFM and 2DICAL enables the comprehensive analysis of plasma proteins and is ideal for use in all biomarker discovery studies. ©2011 AACR.


Yokomizo A.,Kyushu University | Takakura M.,National Cancer Center Research Institute | Kanai Y.,National Cancer Center Research Institute | Sakuma T.,BioBusiness Group | And 5 more authors.
Cancer Biomarkers | Year: 2011

Background: Early detection would be one of the most effective means to improve the outcome of renal cell carcinoma (RCC). We searched for a new plasma marker for RCC using a label-free quantitative shotgun proteomics method. Methods: Plasma proteins were digested by trypsin, and the resulting peptides were analyzed by 2-Dimensional Image Converted Analysis of Liquid chromatography mass spectrometry (2DICAL). An identified biomarker candidate was subjected to validation using the Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA). Results: Among a total of 23,407 independent MS peaks, we found that the mean intensity of 59~peaks significantly differed between 20 clear cell RCC patients and 20~healthy controls. MS/MS spectra from 16 of the 59~peaks matched the amino acid sequences of the fibronectin 1 (FN1) gene product. {The increased plasma level of FN1 in RCC patients was validated in a cohort of in 77 patients and 130 healthy controls (p<0.0001).} Conclusions: The FN1 is considered to be a promising biomarker candidate for clear cell RCC. Furthermore, AlphaLISA is an alternate to the conventional enzyme-linked immunosorbent assay and should prove useful for the rapid validation of biomarker candidates.


Miyanaga A.,National Cancer Center Research Institute | Miyanaga A.,Nippon Medical School | Honda K.,National Cancer Center Research Institute | Tsuta K.,Clinical Pathology Laboratories | And 11 more authors.
Annals of Oncology | Year: 2013

Background: High-grade neuroendocrine tumours (HGNTs) of the lung manifest a wide spectrum of clinical behaviour, butno method for predicting their outcome has been established. Materials and methods: We newly established a monoclonal antibody specifically recognizing the product of the alternatively spliced ACTN4 transcript (namely, variant actinin-4), and used it to examine the expression of variant actinin-4 immunohistochemically in a total of 609 surgical specimens of various histological subtypes of lung cancer. Results: Variant actinin-4 was expressed in 55% (96/176) of HGNTs, but in only 0.8% (3/378) of non-neuroendocrine(NE) lung cancers. The expression of variant actinin-4 was significantly associated with poorer overall survival in HGNT patients (P = 0.00021, log-rank test). Multivariate analysis using the Cox proportional hazards model showed that the expression of variant actinin-4 was the most significant independent negative predictor of survival in HGNT patients(hazard ratio (HR), 2.15; P = 0.00113) after the presence of lymph node metastasis (HR, 2.25; P = 0.00023). Conclusions: The expression of variant actinin-4 is an independent prognostic factor for patients with HGNTs. This protein has a high affinity for filamentous actin polymers and likely promotes aggressive behaviour of cancer cells. The present clinical findings clearly support this notion. © The Author 2012. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved.


Miyanaga A.,National Cancer Center Research Institute | Miyanaga A.,Nippon Medical School | Masuda M.,National Cancer Center Research Institute | Tsuta K.,Clinical Pathology Laboratories | And 6 more authors.
Journal of Thoracic Oncology | Year: 2015

Introduction: Malignant mesothelioma (MM) is an aggressive neoplasm causatively associated with exposure to asbestos. MM is rarely responsive to conventional cytotoxic drugs, and the outcome remains dismal. It is, therefore, necessary to identify the signaling pathways that drive MM and to develop new therapeutics specifically targeting the molecules involved. Methods: We performed comprehensive RNA sequencing of 12 MM cell lines and four clinical samples using so-called next-generation sequencers. Results: We found 15 novel fusion transcripts including one derived from chromosomal translocation between the large tumor suppressor 1 (LATS1) and presenilin-1 (PSEN1) genes. LATS1 is one of the central players of the emerging Hippo signaling pathway. The LATS1-PSEN1 fusion gene product lacked the ability to phosphorylate yes-associated protein and to suppress the growth of a MM cell line. The wild-type LATS1 allele was undetectable in this cell line, indicating two-hit genetic inactivation of its tumor suppressor function. Using pathway-targeted exon sequencing, we further identified a total of 11 somatic mutations in four Hippo pathway genes (neurofibromatosis type 2 [NF2], LATS2, RASSF1, and SAV1) in 35% (8 of 23) of clinical samples. Nuclear staining of yes-associated protein was detected in 55% (24 of 44) of the clinical samples. Expression and/or phosphorylation of the Hippo signaling proteins, RASSF1, Merlin (NF2), LATS1, and LATS2, was frequently absent. Conclusions: The frequent alterations of Hippo pathway molecules found in this study indicate the therapeutic feasibility of targeting this pathway in patients with MM. © 2015 by the International Association for the Study of Lung Cancer.


Honda K.,National Cancer Center Research Institute | Okusaka T.,National Cancer Center Hospital | Felix K.,University of Heidelberg | Nakamori S.,Osaka National Hospital | And 15 more authors.
PLoS ONE | Year: 2012

Background: Among the more common human malignancies, invasive ductal carcinoma of the pancreas has the worst prognosis. The poor outcome seems to be attributable to difficulty in early detection. Methods: We compared the plasma protein profiles of 112 pancreatic cancer patients with those of 103 sex- and age-matched healthy controls (Cohort 1) using a newly developed matrix-assisted laser desorption/ionization (oMALDI) QqTOF (quadrupole time-of-flight) mass spectrometry (MS) system. Results: We found that hemi-truncated apolipoprotein AII dimer (ApoAII-2; 17252 m/z), unglycosylated apolipoprotein CIII (ApoCIII-0; 8766 m/z), and their summed value were significantly decreased in the pancreatic cancer patients [P = 1.36×10-21, P = 4.35×10-14, and P = 1.83×10-24 (Mann-Whitney U-test); area-under-curve values of 0.877, 0.798, and 0.903, respectively]. The significance was further validated in a total of 1099 plasma/serum samples, consisting of 2 retrospective cohorts [Cohort 2 (n = 103) and Cohort 3 (n = 163)] and a prospective cohort [Cohort 4 (n = 833)] collected from 8 medical institutions in Japan and Germany. Conclusions: We have constructed a robust quantitative MS profiling system and used it to validate alterations of modified apolipoproteins in multiple cohorts of patients with pancreatic cancer. © 2012 Honda et al.


Masuda M.,National Cancer Center Research Institute | Chen W.-Y.,Taipei Medical University | Miyanaga A.,National Cancer Center Research Institute | Nakamura Y.,National Cancer Center Research Institute | And 6 more authors.
Molecular and Cellular Proteomics | Year: 2014

Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.


Kobayashi E.,National Cancer Center Research Institute | Kobayashi E.,Keio University | Masuda M.,National Cancer Center Research Institute | Nakayama R.,Genetics Division | And 14 more authors.
Molecular Cancer Therapeutics | Year: 2010

Pulmonary metastasis is the most significant prognostic determinant for osteosarcoma, but methods for its prediction and treatment have not been established. Using oligonucleotide microarrays, we compared the global gene expression of biopsy samples between seven osteosarcoma patients who developed pulmonary metastasis within 4 years after neoadjuvant chemotherapy and curative resection, and 12 patients who did not relapse.We identified argininosuccinate synthetase (ASS) as a gene differentially expressed with the highest statistical significance (Welch's t test, P = 2.2 × 10-5). Immunohistochemical analysis of an independent cohort of 62 osteosarcoma cases confirmed that reduced expression of ASS protein was significantly correlated with the development of pulmonary metastasis after surgery (log-rank test, P < 0.05). Cox regression analysis revealed that ASS was the sole significant predictive factor (P = 0.039; hazard ratio, 0.319; 95% confidence interval, 0.108-0.945). ASS is one of the enzymes required for the production of a nonessential amino acid, arginine. We showed that osteosarcoma cells lacking ASS expression were auxotrophic for arginine and underwent G0-G 1 arrest in arginine-free medium, suggesting that an arginine deprivation therapy could be effective in patients with osteosarcoma. Recently, phase I and II clinical trials in patients with melanoma and hepatocellular carcinoma have shown the safety and efficacy of plasma arginine depletion by stabilized arginine deiminase. Our data indicate that in patients with osteosarcoma, reduced expression of ASS is not only a novel predictive biomarker for the development of metastasis, but also a potential target for pharmacologic intervention. ©2010 American Association for Cancer Research.


Kobayashi E.,National Cancer Center Research Institute | Kobayashi E.,Keio University | Satow R.,National Cancer Center Research Institute | Ono M.,National Cancer Center Research Institute | And 7 more authors.
Oncology (Switzerland) | Year: 2014

Objective: Osteosarcoma (OS) is the most frequent primary malignant bone tumor in children and young adults. Although the introduction of combined neoadjuvant chemotherapy has significantly prolonged survival, the outcome for OS patients showing a poor response to chemotherapy is still unfavorable. In order to develop new therapeutic approaches, elucidation of the entire molecular pathway regulating OS cell proliferation would be desirable. Methods: MicroRNA (miRNA) are highly conserved noncoding RNA that play important roles in the development and progression of various other cancers. Using miRNA microarrays capable of detecting a known number of 933 miRNA, 108 miRNA were found to be commonly expressed in 24 samples of OS tissue and subjected to a cell proliferation assay. Results: We found that inhibition of 5 let-7 family miRNA (hsa-let-7a, b, f, g and i) significantly suppressed the proliferation of OS cells. Using a quantitative shotgun proteomics approach, we also found that the let-7 family miRNA regulated the expression of vimentin and serpin H1 proteins. Conclusions: Our present results indicate the involvement of let-7 family miRNA in regulation of the cell proliferation as well as epithelial-mesenchymal transition of OS. Thus, let-7 family miRNA may potentially provide novel targets for the development of therapeutic strategies against OS. © 2014 S. Karger AG, Basel.

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