Background. Idiopathic pure red cell aplasia (PRCA) and secondary PRCA not responding to the treatment of the underlying diseases are generally thought be immune-mediated and treated by ...immunosuppressive therapy. We previously conducted the PRCA2004/2006 study and reported that poor response to induction therapy and relapse of anemia were associated with death. Principal causes of death were infections and organ failure. Based on the literatures, idiopathic PRCA may represents the prodrome to myelodysplastic syndromes. Theoretically, there are two potential mechanisms of unresponsiveness to immunosuppression; the clonal hematopoiesis by the stem/progenitor cells that have undergone somatic mutations during disease progression of PRCA and the clonal changes of auto-aggressive lymphocytes reacting against erythroid progenitors.
Objectives. In this study, we investigated the somatic mutations of myeloid malignancy-associated genes in acquired PRCA in order to determine how often clonal hematopoiesis is detected in this disorder.
Materials and Methods. This study included 23 patients with chronic acquired PRCA (12 idiopathic, 7 thymoma-, 2 LGL leukemia- and 2 systemic lupus erythematosus-associated PRCA) with a median age of 62 (range: 40-62). Disease status was varying. After obtaining informed consent, heparinized blood was drawn and mononuclear cells were separated by density gradient centrifugation. Extracted genomic DNA samples were subjected to targeted sequencing for 54 myeloid malignancy-associated genes using a TruSight Myeloid Sequencing Panel kit according to the manufacturer's instruction (Illumina). Criteria for the significant somatic mutations of myeloid malignancy-associated genes in the present study were as follows: potential functional consequences such as missense, nonsense or frameshift mutations; exclusion of previously reported SNPs; being recurrently detected in two sequencing runs; variant allele frequency (VAF) exceeding 0.02 and less than 0.40. The institutional review board approved the experimental protocol.
Results. We detected some mutations of the targeted genes in 20 out of 23 patients, and the somatic mutations defined by the criteria mentioned above were found in 10 patients including 6 idiopathic, 3 thymoma-associated and one LGL leukemia-associated PRCA (Fig. 1). These 10 patients had 38 distinct mutations in 20 genes. Variant allele frequencies were 0.02 to 0.37 (median, 0.04; average, 0.06, Fig. 2). Four patients had more than one mutated genes and multiple genes were mutated in some patients (Fig. 1). The most frequently mutated gene was CUX1 that was found in four patients, and STAG2, DNMT3A, KDM6A, SMC3A, ASXL1, TET2 and TP53 were mutated in more than one patient.
Discussion/Conclusion. This study demonstrated that myeloid malignancy-associated genes were somatically mutated in 43% of acquired chronic PRCA patients. This figure appears to exceed the prevalence rate of clonal hematopoiesis of indeterminate potential (CHIP) in the general population with the age of 60s. These mutations were presumably carried by monocytes, because DNA samples were prepared from PBMCs in this study cohort. Profiles of mutated genes in PRCA appear to be different from those of aplastic anemia that were previously reported by other groups. It is yet to be known whether this could result from the different nature of both diseases, or the difference in the experimental protocols. Our findings strongly encourage conducting a prospective study to confirm our observation and clarify the diagnostic and predictive values of somatic mutations of myeloid malignancy-associated genes in acquired PRCA. This project is ongoing in collaboration with the prospective cohort study PRCA2016 being conducted in Japan.
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Nakao:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Matsuda:GlaxoSmithKline K.K.: Honoraria; Novartis Pharma K. K.: Honoraria; Chugai Pharmaceutical Co, Ltd.: Honoraria; Kyowa Hakko Kirin Co, Ltd.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria; Celgene Corporation: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Sanofi K.K.: Honoraria; Beckman Coulter K.K.: Honoraria. Mitani:Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau; Bristol-Myesr Squibb: Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Chugai: Research Funding; Astellas: Research Funding; Sumitomo Dainippon: Research Funding; Novartis: Research Funding; Toyama Chemical: Research Funding.
Identification of the key molecules that mediate susceptibility to anticancer treatments would be highly desirable. Based on clinical and cell biological studies, we recently proposed that ...regenerating gene (REG) Iα may be such a molecule. In the present study, we hypothesized that REG Iα increases radiosensitivity through activation of mitogen-activated protein kinase (MAPK) pathways. To test that idea, we transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Iα and examined its involvement in MAPK signaling and its effect on susceptibility to radiotherapy. We found that REG Iα-expressing cells showed increased expression of c-Jun messenger RNA (mRNA) and phospho-c-Jun protein mediated via the c-Jun N-terminal kinase (JNK) pathway and extracellular signal-regulated kinase (ERK) pathway, as well as increased radiosensitivity. Immunohistochemical analysis confirmed the activation of c-Jun in tumors expressing REG Iα. Collectively, these findings suggest that REG Iα activates c-Jun via the JNK and ERK pathway, thereby enhancing radiosensitivity.
Background Transforming growth factor (TGF)-beta is known to be produced by progressor tumors and to immobilize dendritic cells (DCs) within those tumors. Moreover, although TGF-beta1 has been shown ...to promote tumor progression, there is still no direct, in vivo evidence as to whether TGF-beta1 is able to directly induce distant metastasis. Methods To address that issue and investigate the mechanism by which TGF-beta1 suppresses DC activity, we subdermally inoculated mouse ears with squamous cell carcinoma cells stably expressing TGF-beta1 or empty vector (mock). Results The numbers of DCs within lymph nodes draining the resultant TGF-beta1-expressing tumors was significantly lower than within nodes draining tumors not expressing TGF-beta1. We then injected fluorescently labeled bone marrow-derived dendritic cells into the tumors, and subsequent analysis confirmed that the tumors were the source of the DCs within the tumor-draining lymph nodes, and that there were significantly fewer immature DCs within the nodes draining TGF-beta1-expressing tumors than within nodes draining tumors not expressing TGF-beta1. In addition, 14 days after tumor cell inoculation, lymph node metastasis occurred more frequently in mice inoculated with TGF-beta1 transfectants than in those inoculated with the mock transfectants. Conclusions These findings provide new evidence that tumor-derived TGF-beta1 inhibits migration of DCs from tumors to their draining lymph nodes, and this immunosuppressive effect of TGF-beta1 increases the likelihood of metastasis in the affected nodes.
Identification of the key molecules that mediate susceptibility to anticancer treatments would be highly desirable. Based on clinical and cell biological studies, we recently proposed that ...regenerating gene (REG) Ialpha may be such a molecule. In the present study, we hypothesized that REG Ialpha increases radiosensitivity through activation of mitogen-activated protein kinase (MAPK) pathways. To test that idea, we transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Ialpha and examined its involvement in MAPK signaling and its effect on susceptibility to radiotherapy. We found that REG Ialpha-expressing cells showed increased expression of c-Jun messenger RNA (mRNA) and phospho-c-Jun protein mediated via the c-Jun N-terminal kinase (JNK) pathway and extracellular signal-regulated kinase (ERK) pathway, as well as increased radiosensitivity. Immunohistochemical analysis confirmed the activation of c-Jun in tumors expressing REG Ialpha. Collectively, these findings suggest that REG Ialpha activates c-Jun via the JNK and ERK pathway, thereby enhancing radiosensitivity.
Genet. 46, 722-725 (2014); published online 25 May 2014; corrected after print 12 November 2014 In the version of this article initially published, the introductory paragraph mistakenly stated that ...ameloblasts were "cells in the tooth roots of the upper (maxilla) and lower (mandible) jaw responsible for depositing enamel during tooth development (odontogenesis)."
β1,4‐N‐Acetylglucosaminyltransferase III (GnT‐III) plays a regulatory role in the biosynthesis of N‐glycans, and it has been suggested that its product, a bisecting GlcNAc, is involved in a variety ...of biological events as well as in regulating the biosynthesis of the oligosaccharides. In this study, it was found, on the basis of sequence homology, that GnT‐III contains a small region that is significantly homologous to both snail β1,4GlcNAc transferase and β1,4Gal transferase‐1. Subsequent mutational analysis demonstrated an absolute requirement for two conserved Asp residues (Asp321 and Asp323), which are located in the most homologous region of rat GnT‐III, for enzymatic activity. The overexpression of Asp323‐substituted, catalytically inactive GnT‐III in Huh6 cells led to the suppression of the activity of␣endogenous GnT‐III, but no significant decrease in its expression, and led to a specific inhibition of the formation of bisected sugar chains, as shown by structural analysis of the total N‐glycans from the cells. These findings indicate that the mutant serves a dominant negative effect on a specific step in N‐glycan biosynthesis. This type of ‘dominant negative glycosyltransferase’, identified has potential value as a powerful tool for defining the precise biological roles of the bisecting GlcNAc structure.
Regenerating gene (REG) I plays important roles in cancer cell biology. The purpose of this study was to determine whether REG I affects cytokine production in cancer cells. We transfected TE-5 and ...TE-9 squamous esophageal cancer cells with REG Iα and Iβ and examined its effects on cytokine expression. We found that transfecting TE-5 and TE-9 cells with REG I Iα and Iβ led to significantly increased expression of interleukin (IL)-6 mRNA and protein, but it had little or no effect on expression of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17A, interferon-γ, tumor necrosis factor-α, granulocyte-colony stimulating factor or transforming growth factor-β1. The elevated IL-6 expression seen in REG Iα transfectants was silenced by small interfering RNA-mediated knockdown. These finding suggest that REG I may act through IL-6 to exert effects on squamous esophageal cancer cell biology.
Background
A reliable marker of chemoradiosensitivity that would enable appropriate and individualized treatment of thoracic squamous cell esophageal cancer has long been sought. We investigated ...whether regenerating gene (REG) Iα is such a marker.
Methods
We assessed expression of REG Iα in untreated endoscopic biopsy specimens and examined the correlation between REG Iα expression and the clinical responses to definitive chemoradiotherapy and prognosis. We also examined the relationship between REG Iα expression in the resected tumor and the prognosis of patients who received esophagectomy for thoracic squamous cell esophageal cancer.
Results
Among the 42 patients treated with definitive chemoradiotherapy, 8 of the 23 REG I-positive patients (35%) showed complete responses to chemoradiotherapy, while only one of the 19 REG I-negative patients did so. The survival rate among the REG I-positive patients was significantly better than among the REG I-negative patients. For the 76 patients treated surgically, there was no significant difference in the survival rates among the REG I-positive and REG I-negative patients.
Conclusions
REG Iα expression in squamous cell esophageal carcinoma may be a reliable marker of chemoradiosensitivity. We anticipate that it will enable us to provide more appropriate and individualized treatment to patients of advanced esophageal squamous cell carcinoma.
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