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Niikura N.,Tokai University | Niikura N.,Study Group of Standard Measurement of Ki67 | Sakatani T.,Study Group of Standard Measurement of Ki67 | Sakatani T.,Jichi Medical University | And 17 more authors.
Breast Cancer | Year: 2016

Background: A lack of consistent methods to evaluate Ki67 expression is problematic in terms of accurately predicting prognosis in breast cancer. Accordingly, this study aimed to identify the causes of discrepancies in Ki67 labeling index measurements by different observers under different conditions using breast cancer samples. Patients and methods: This Japanese study group compared and assessed immunohistochemical (IHC) analysis of the Ki67 labeling index when measured by different pathologists. Six pathologists (pathologists A–F) in Japan participated in this ring study. One hundred and ten surgical cases of estrogen receptor-positive and human epidermal growth factor receptor 2-negative invasive breast cancer treated in 2007 were identified from the breast cancer database of Tokai University Hospital and were included in this study. Results: For all 6 pathologists, the Ki67 labeling index were significantly different between grade 3 and grade 1 cases and between grade 3 and grade 2 cases, whereas the index tended to be different between grade 1 and grade 2 cases. Further, the Ki67 labeling indexes measured by the 6 pathologists were strongly correlated (ρ: 0.73–0.88). The IHC scores recorded by pathologist A were in moderate to good agreement with those recorded by the others in patients with a Ki67 labeling index of <13.25 % and in those with a Ki67 labeling index of >13.25 % (κ = 0.429–0.660). The Ki67 low and high concordance rates between pathologist A and the others were 0.452–0.778 and 0.862–0.979, respectively. The most pertinent reason for discrepancy in scores seemed to be the selection of the area for counting and the quality of nuclear staining. Conclusion: The Ki67 labeling index measured by 6 pathologists without method standardization was in fair to good agreement. We plan to undertake a second ring study, pending recommendations by the international Ki67 panel. © 2014, The Japanese Breast Cancer Society. Source

Hirata O.,Hiroshima University | Okada S.,Hiroshima University | Tsumura M.,Hiroshima University | Karakawa S.,Hiroshima University | And 7 more authors.
Journal of Clinical Immunology | Year: 2015

Purpose: To confirm and characterize mosaicism of the cyclic neutropenia (CyN)-related mutation in the ELANE gene identified in the asymptomatic mother of patients with CyN. Methods: We identified sibling cases with CyN due to a novel heterozygous splicing site mutation, IVS4 +5SD G>T, in the ELANE gene, resulting in an internal in-frame deletion of 30 nucleotides (corresponding to a ten amino acid deletion, V161–F170). The mutated allele was also detected in their asymptomatic mother but at low frequency. We measured the frequency of the mutant allele from peripheral blood leukocytes (PBLs) by subcloning, and confirmed the allelic frequency of mosaicism in various cell types by massively parallel DNA sequencing (MPS) analysis. Results: In the subcloning analysis, the mutant allele was identified in 21.36 % of PBLs from the asymptomatic mother, compared with 54.72 % of PBLs from the CyN patient. In the MPS analysis, the mutant allele was observed in approximately 30 % of mononuclear cells, CD3+ T cells, CD14+ monocytes and the buccal mucosa. Conversely, it was detected in low frequency in polymorphonuclear leukocytes (PLMLs) (3–4 %) and CD16+ granulocytes (2–3 %). Conclusions: Mosaicism of the ELANE mutation has only previously been identified in one confirmed and one unconfirmed case of SCN. This is the first report of mosaicism of the ELANE mutation in a case of CyN. The MPS results suggest that this de novo mutation occurred during the two-cell stage of embryogenesis. PLMLs expressing the ELANE mutation were found to be actively undergoing apoptosis. © 2015, Springer Science+Business Media New York. Source

Mimae T.,Research Institute for Radiation Biology and Medicine | Mimae T.,Japan Institute for Molecular Science | Hagiyama M.,Kinki University | Inoue T.,Kinki University | And 5 more authors.
Thorax | Year: 2014

Rationale: Alveolar epithelial cell apoptosis and protease/antiprotease imbalance based proteolysis play central roles in the pathogenesis of pulmonary emphysema but molecular mechanisms underlying these two events are not yet clearly understood. Cell adhesion molecule 1 (CADM1) is a lung epithelial cell adhesion molecule in the immunoglobulin superfamily. It generates two membrane associated C terminal fragments (CTFs), αCTF and βCTF, through protease mediated ectodomain shedding. Objective: To explore the hypothesis that more CADM1-CTFs are generated in emphysematous lungs through enhanced ectodomain shedding, and cause increased apoptosis of alveolar epithelial cells. Methods and results: Western blot analyses revealed that CADM1-CTFs increased in human emphysematous lungs in association with increased ectodomain shedding. Increased apoptosis of alveolar epithelial cells in emphysematous lungs was confirmed by terminal nucleotide nick end labelling (TUNEL) assays. NCI-H441 lung epithelial cells expressing mature CADM1 but not CTFs were induced to express αCTF both endogenously (by shedding inducers phorbol ester and trypsin) and exogenously (by transfection). Cell fractionation, immunofluorescence, mitochondrial membrane potentiometric JC-1 dye labelling and TUNEL assays revealed that CADM1-αCTF was localised to mitochondria where it decreased mitochondrial membrane potential and increased cell apoptosis. A mutation in the intracytoplasmic domain abrogated all three abilities of αCTF. Conclusions: CADM1 ectodomain shedding appeared to cause alveolar cell apoptosis in emphysematous lungs by producing αCTF that accumulated in mitochondria. These data link proteolysis to apoptosis, which are two landmark events in emphysema. Source

Hosoya N.,University of Tokyo | Okajima M.,University of Tokyo | Kinomura A.,Research Institute for Radiation Biology and Medicine | Kinomura A.,Research Institute for Radiation Biology and Medicine | And 6 more authors.
EMBO Reports | Year: 2012

The meiosis-specific synaptonemal complex protein SYCP3 has been reported to be aberrantly expressed in tumours. However, in contrast to its well-defined function in meiosis, its possible role in mitotic cells is entirely unknown. Here, we show that SYCP3 is expressed in a range of primary tumours and that it impairs chromosomal integrity in mitotic cells. Expression of SYCP3 inhibits the homologous recombination (HR) pathway mediated by RAD51, inducing hypersensitivity to DNA-damaging agents such as a poly(ADP-ribose) polymerase (PARP) inhibitor and chromosomal instability. SYCP3 forms a complex with BRCA2 and inhibits its role in HR. These findings highlight a new mechanism for chromosomal instability in cancer and extend the range of PARP-inhibitor sensitive tumours to those expressing SYCP3. © 2012 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION. Source

Morino H.,Research Institute for Radiation Biology and Medicine | Pierce S.B.,University of Washington | Matsuda Y.,Research Institute for Radiation Biology and Medicine | Walsh T.,University of Washington | And 11 more authors.
Neurology | Year: 2014

Objective: To identify the genetic cause in 2 families of progressive ataxia, axonal neuropathy, hyporeflexia, and abnormal eye movements, accompanied by progressive hearing loss and ovarian dysgenesis, with a clinical diagnosis of Perrault syndrome. Methods: Whole-exome sequencing was performed to identify causative mutations in the 2 affected sisters in each family. Family 1 is of Japanese ancestry, and family 2 is of European ancestry. Results: In family 1, affected individuals were compound heterozygous for chromosome 10 open reading frame 2 (C10orf2) p.Arg391His and p.Asn585Ser. In family 2, affected individuals were compound heterozygous for C10orf2 p.Trp441Gly and p.Val507Ile. C10orf2 encodes Twinkle, a primase-helicase essential for replication of mitochondrial DNA. Conservation and structural modeling support the causality of the mutations. Twinkle is known also to harbor multiple mutations, nearly all missenses, leading to dominant progressive external ophthalmoplegia type 3 and to recessive mitochondrial DNA depletion syndrome 7, also known as infantile-onset spinocerebellar ataxia. Conclusion: Our study identifies Twinkle mutations as a cause of Perrault syndrome accompanied by neurologic features and expands the phenotypic spectrum of recessive disease caused by mutations in Twinkle. The phenotypic heterogeneity of conditions caused by Twinkle mutations and the genetic heterogeneity of Perrault syndrome call for genomic definition of these disorders. © 2014 American Academy of Neurology. Source

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