Publications by authors named "Yusuke Nakamura"

926 Publications

Genetic variations in medical research in the past, at present and in the future.

Proc Jpn Acad Ser B Phys Biol Sci 2021 ;97(6):324-335

Cancer Precision Medicine Center, Japanese Foundation for Cancer Research.

As we look so different, our genomic sequences vary enormously. The differences in our genome, genetic variations, have played very significant roles in medical research and have contributed to improvement of medical managements in the last 2-3 decades. Genetic variations include germline variations, somatic mutations, and diversities in receptor genes of rearranged immune cells, T cells and B cells. Germline variants are in some cases causative of genetic diseases, are associated with the risk of various diseases, and also affect drug efficacies or adverse events. Some somatic mutations are causative of tumor development. Recent DNA sequencing technologies allow us to perform single-cell analysis or detailed repertoire analysis of B and T cells. It is critically important to investigate temporal changes in immune environment in various anatomical regions in the next one to two decades. In this review article, we would like to introduce the roles of genetic variations in medical fields in the past, at present and in the future.
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http://dx.doi.org/10.2183/pjab.97.018DOI Listing
January 2021

Iatrogenic Middle Meningeal Arteriovenous Fistula During Embolization: Two Case Reports and Literature Review.

J Stroke Cerebrovasc Dis 2021 Jun 10;30(8):105909. Epub 2021 Jun 10.

Department of Neurosurgery, St. Mary's Hospital, 422 Tsubukuhonnmachi, Kurume, Fukuoka 8308543, Japan. Electronic address:

Objective Dural arteriovenous fistulae of the middle meningeal artery are rare. There are few reports of complications associated with endovascular therapy. This report describes two cases of iatrogenic middle meningeal arteriovenous fistula due to vascular injury sustained during endovascular treatment. Case description Case 1 was that of a 46-year-old woman. She was treated for an incidentally discovered dural arteriovenous fistula of the cerebellar tentorium by transarterial embolization. During the procedure, a middle meningeal arteriovenous fistula occurred because of vessel laceration by the forced advancement of the distal access catheter (DAC). After the intervention, she developed tinnitus. Follow-up angiography revealed a middle meningeal arteriovenous fistula. The fistula was treated by coil embolization of the affected middle meningeal artery. The second case was that of a 56-year-old woman who developed a middle meningeal arteriovenous fistula from the perforation caused by the microguidewire during tumor embolization. The fistula was treated by occluding the proximal segment of the affected artery with coils. Both patients were discharged without neurological complications after the endovascular procedures. Conclusion Endovascular surgeons should be aware of the possibility of middle meningeal arteriovenous fistula as a potential complication of endovascular procedures.
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http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2021.105909DOI Listing
June 2021

Generation of neoantigen-specific T cells for adoptive cell transfer for treating head and neck squamous cell carcinoma.

Oncoimmunology 2021 May 25;10(1):1929726. Epub 2021 May 25.

Cytotherapy Laboratory, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; the First Affiliated Hospital, Southern University of Science and Technology), Shenzhen Guangdong, China.

Adoptive cell therapy using TCR-engineered T cells (TCR-T cells) represents a promising strategy for treating relapsed and metastatic cancers. We previously established methods to identify neoantigen-specific TCRs based on patients' PBMCs. However, in clinical practice isolation of PBMCs from advanced-stage cancer patients proves to be difficult. In this study, we substituted blood-derived T cells for tumor-infiltrating lymphocytes (TILs) and used an HLA-matched cell line of antigen-presenting cells (APCs) to replace autologous dendritic cells. Somatic mutations were determined in head and neck squamous cell carcinoma resected from two patients. HLA-A*02:01-restricted neoantigen libraries were constructed and transferred into HLA-matched APCs for stimulation of patient TILs. TCRs were isolated from reactive TIL cultures and functionality was tested using TCR- T cells and . To exemplify the screening approach, we identified the targeted neoantigen leading to recognition of the minigene construct that stimulated the strongest TIL response. Neoantigen peptides were used to load MHC-tetramers for T cell isolation and a TCR was identified targeting the KIAA1429 mutation. TCR-T cells were activated, exhibited cytotoxicity, and secreted cytokines in a dose-dependent manner, and only when stimulated with the mutant peptide. Furthermore, comparable to a neoantigen-specific TCR that was isolated from the patient's PBMCs, KIAA1429-specific TCR T cells destroyed human tumors in mice. The established protocol provides the required flexibility to methods striving to identify neoantigen-specific TCRs. By using an MHC-matched APC cell line and neoantigen-encoding minigene libraries, autologous TILs can be stimulated and screened when patient PBMCs and/or tumor material are not available anymore. Abbreviations: Head and neck squamous cell carcinoma (HNSCC); adoptive T cell therapy (ACT); T cell receptor (TCR); tumor-infiltrating lymphocytes (TIL); cytotoxic T lymphocyte (CTL); peripheral blood mononuclear cell (PBMC); dendritic cell (DC); antigen-presenting cells (APC).
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http://dx.doi.org/10.1080/2162402X.2021.1929726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158031PMC
May 2021

Clinical implementation and current advancement of blood liquid biopsy in cancer.

J Hum Genet 2021 Jun 4. Epub 2021 Jun 4.

Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

Liquid biopsies have been receiving tremendous attentions as easy, rapid, and non-invasive tools for cancer diagnosis. Liquid biopsy can be performed repeatedly for disease monitoring and is expected to overcome the limitations of tissue biopsies. With the advancement of next generation sequencing technologies, it is now possible to detect minute amount of tumor-derived circulation tumor DNA (ctDNA) from blood samples. Importantly, ctDNA detection could be complementary to tissue biopsies or tumor biomarkers particularly in cases of which tumor biopsy is clinically difficult to obtain. Here, we introduce the up-to-date technologies used in cfDNA-based liquid biopsy and review the clinical utilities of ctDNA in cancer screening, detection of minimal residual diseases, selection of molecular-targeted drugs, as well as monitoring of treatment responsiveness. We also discuss the challenges and future perspectives of liquid biopsy implementation in clinical setting.
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http://dx.doi.org/10.1038/s10038-021-00939-5DOI Listing
June 2021

Highly Selective Synthesis of α-Aminoamide Utilizing an Umpolung Reaction and Characteristics of α-Hydrazonoester.

Org Lett 2021 Jun 20;23(11):4168-4172. Epub 2021 May 20.

Department of Chemistry for Materials, Graduate School of Engineering, Mie University, Tsu, Mie 514-8507, Japan.

An umpolung reaction with α-hydrazonoesters was investigated, and it was found that α-,-dialkylaminoamides could be directly synthesized in yields up to 92% via a concomitant rearrangement of dialkylamino groups. As an application, a short synthesis of an inhibitor of glycine type-1-transporter was accomplished via subsequent functional group transformations in 28% overall yield.
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http://dx.doi.org/10.1021/acs.orglett.1c01117DOI Listing
June 2021

Application of targeted nanopore sequencing for the screening and determination of structural variants in patients with Lynch syndrome.

J Hum Genet 2021 May 6. Epub 2021 May 6.

Division of Clinical Genome Research, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

Lynch syndrome is a hereditary disease characterized by an increased risk of colorectal and other cancers. Germline variants in the mismatch repair (MMR) genes are responsible for this disease. Previously, we screened the MMR genes in colorectal cancer patients who fulfilled modified Amsterdam II criteria, and multiplex ligation-dependent probe amplification (MPLA) identified 11 structural variants (SVs) of MLH1 and MSH2 in 17 patients. In this study, we have tested the efficacy of long read-sequencing coupled with target enrichment for the determination of SVs and their breakpoints. DNA was captured by array probes designed to hybridize with target regions including four MMR genes and then sequenced using MinION, a nanopore sequencing platform. Approximately, 1000-fold coverage was obtained in the target regions compared with other regions. Application of this system to four test cases among the 17 patients correctly mapped the breakpoints. In addition, we newly found a deletion across an 84 kb region of MSH2 in a case without the pathogenic single nucleotide variants. These data suggest that long read-sequencing combined with hybridization-based enrichment is an efficient method to identify both SVs and their breakpoints. This strategy might replace MLPA for the screening of SVs in hereditary diseases.
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http://dx.doi.org/10.1038/s10038-021-00927-9DOI Listing
May 2021

Genome-wide association study of epilepsy in a Japanese population identified an associated region at chromosome 12q24.

Epilepsia 2021 Jun 29;62(6):1391-1400. Epub 2021 Apr 29.

Department of Neurodevelopmental Disorder Genetics, Institute of Brain Science, Nagoya City University Graduate School of Medical Science, Aichi, Japan.

Objective: Although a number of genes responsible for epilepsy have been identified through Mendelian genetic approaches, and genome-wide association studies (GWASs) have implicated several susceptibility loci, the role of ethnic-specific markers remains to be fully explored. We aimed to identify novel genetic associations with epilepsy in a Japanese population.

Methods: We conducted a GWAS on 1825 patients with a variety of epilepsies and 7975 control individuals. Expression quantitative trait locus (eQTL) analysis of epilepsy-associated single nucleotide polymorphisms (SNPs) was performed using Japanese eQTL data.

Results: We identified a novel region, which is ~2 Mb (lead SNP rs149212747, p = 8.57 × 10 ), at chromosome 12q24 as a risk for epilepsy. Most of these loci were polymorphic in East Asian populations including Japanese, but monomorphic in the European population. This region harbors 24 transcripts including genes expressed in the brain such as CUX2, ATXN2, BRAP, ALDH2, ERP29, TRAFD1, HECTD4, RPL6, PTPN11, and RPH3A. The eQTL analysis revealed that the associated SNPs are also correlated to differential expression of genes at 12q24.

Significance: These findings suggest that a gene or genes in the CUX2-RPH3A ~2-Mb region contribute to the pathology of epilepsy in the Japanese population.
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http://dx.doi.org/10.1111/epi.16911DOI Listing
June 2021

Coordination sequences of crystals are of quasi-polynomial type.

Acta Crystallogr A Found Adv 2021 Mar 18;77(Pt 2):138-148. Epub 2021 Feb 18.

Graduate School of Mathematical Sciences, University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo, 153-8914, Japan.

The coordination sequence of a graph measures how many vertices the graph has at each distance from a fixed vertex and is a generalization of the coordination number. Here it is proved that the coordination sequence of the graph obtained from a crystal is of quasi-polynomial type, as had been postulated by Grosse-Kunstleve et al. [Acta Cryst. (1996), A52, 879-889].
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http://dx.doi.org/10.1107/S2053273320016769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941273PMC
March 2021

Anti-cancer immunotherapy using cancer-derived multiple epitope-peptides cocktail vaccination clinical studies in patients with refractory/persistent disease of uterine cervical cancer and ovarian cancer [phase 2].

Oncoimmunology 2020 11 11;9(1):1838189. Epub 2020 Nov 11.

Department of Cancer, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

We had conducted phase 1/2 studies of cancer vaccination therapy using neo-tumor antigens in patients with refractory/persistent cervical cancer (CC) and ovarian cancer (OC) to assess the feasibility and efficacy. Enrollees must be refractory/persistent disease for usual treatments with Human Leukocyte Antigen-A*0201 or A*2402. The targets were epitope peptides obtained from driver genes in surviving pathways as follows: for CC A*0201, peptides from Up Regulating Lung Cancer 10 gene (URLC10) and Hypoxia-inducible gene 2 (HIG-2) and for OC A*0201, HIG2, VEGFR (vascular epithelial growth factor receptor) 1 and 2 were used. For CC A*2402, Forkhead Box M1 (FOXM1), Maternal Embryonic Leucine zipper Kinase (MELK), and Holliday Junction Recognition Protein (HJURP) were used. For OC A*2402, cocktails of peptides from FOXM1, MELK, HJURP, VEGFR1, and VEGFR2 were used. Subcutaneous administration was performed with adjuvant weekly. The toxicity profiles and tumor-response were analyzed in eight-week interval. Sixty-six patients were accrued, and 64 were evaluable for adverse events (AEs), and 35 for response. AEs of G2/3 dermatologic reaction (DR) of injection site had been identified in 15.6% and no other severe AEs were detected. Response rate in OC and CC were 22.9% and 20%, respectively. Median overall survival showed longer in performance status (PS) 0 (versus PS1/2), in CRP negative (versus positive) and in DR positive (versus negative) such as 8.7 m versus 1.2 m ( < .001), 8.8 m versus 3.0 m ( < .05) and 10.2 m versus 1.2 m ( < .001), respectively. In conclusion, our vaccination therapy was feasible and effective in this cohort of patients.
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http://dx.doi.org/10.1080/2162402X.2020.1838189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671072PMC
November 2020

Correction: Preclinical evaluation of biomarkers associated with antitumor activity of MELK inhibitor.

Oncotarget 2020 Oct 13;11(41):3749-3750. Epub 2020 Oct 13.

Department of Medicine and Surgery, The University of Chicago, Chicago, IL, USA.

[This corrects the article DOI: 10.18632/oncotarget.7685.].
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http://dx.doi.org/10.18632/oncotarget.27755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566805PMC
October 2020

Established fibrous peritoneal metastasis in an immunocompetent mouse model similar to clinical immune microenvironment of gastric cancer.

BMC Cancer 2020 Oct 20;20(1):1014. Epub 2020 Oct 20.

Department of Gastroenterological Surgery, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa, 920-8641, Japan.

Background: Peritoneal metastasis (PM) in gastric cancer (GC) is characterized by diffusely infiltrating and proliferating cancer cells accompanied by extensive stromal fibrosis in the peritoneal space. The prognosis of GC with PM is still poor regardless of the various current treatments. In order to elucidate the cause of difficulties in PM treatment, we compared the tumor immune microenvironment (TME) in primary and PM lesions in GC. In addition, a PM model with fibrous stroma was constructed using immunocompetent mice to determine whether its TME was similar to that in patients.

Methods: Immuno-histochemical analyses of infiltrating immune cells were performed in paired primary and PM lesions from 28 patients with GC. A C57BL/6 J mouse model with PM was established using the mouse GC cell line YTN16 either with or without co-inoculation of mouse myofibroblast cell line LmcMF with α-SMA expression. The resected PM from each mouse model was analyzed the immunocompetent cells using immunohistochemistry.

Results: The number of CD8 cells was significantly lower in PM lesions than in primary lesions (P < 0.01). Conversely, the number of CD163 cells (M2 macrophages) was significantly higher in PM lesions than in primary lesions (P = 0.016). Azan staining revealed that YTN16 and LmcMF co-inoculated tumors were more fibrous than tumor with YTN16 alone (P < 0.05). Co-inoculated fibrous tumor also showed an invasive growth pattern and higher progression than tumor with YTN16 alone (P = 0.045). Additionally, YTN16 and LmcMF co-inoculated tumors showed lower infiltration of CD8 cells and higher infiltration of M2 macrophages than tumors with YTN16 alone (P < 0.05, P < 0.05). These results indicate that LmcMF plays as cancer-associated fibroblasts (CAFs) by crosstalk with YTN16 and CAFs contribute tumor progression, invasion, fibrosis, and immune suppression.

Conclusions: This model is the first immunocompetent mouse model similar to TME of human clinical PM with fibrosis. By using this model, new treatment strategies for PM, such as anti-CAFs therapies, may be developed.
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http://dx.doi.org/10.1186/s12885-020-07477-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574408PMC
October 2020

High expression of maternal embryonic leucine-zipper kinase (MELK) impacts clinical outcomes in patients with ovarian cancer and its inhibition suppresses ovarian cancer cells growth ex vivo.

J Gynecol Oncol 2020 Nov;31(6):e93

Department of Gynecologic Oncology, Saitama Medical University International Medical Center, Hidaka, Japan.

Objective: Maternal embryonic leucine zipper kinase (MELK) is receiving an attention as a therapeutic target in various types of cancers. In this study, we aimed to evaluate the prognostic significance of expression in ovarian cancer using clinical samples, and assessed the efficacy of a small molecule MELK inhibitor, OTS167, using patient-derived ovarian cancer cells as well as cell lines.

Methods: Expression levels of MELK in 11 ovarian cancer cell lines were confirmed by western blotting. Inhibitory concentration of OTS167 was determined by colorimetric assay. messenger RNA (mRNA) expression was evaluated in 228 ovarian cancer patients by quantitative polymerase chain reaction. Growth inhibition of OTS167 was also evaluated using freshly-isolated primary ovarian cancer cells including spheroid formation condition.

Results: mRNA expression was significantly higher in ovarian cancer than in normal ovaries (p<0.001), and high mRNA expression was observed in patients with advanced stage, positive ascites cytology and residual tumor size. Patients with high mRNA expression showed shorter progression-free survival (p=0.001). Expression of MELK was also confirmed in 10 of 11 ovarian cancer cell lines tested, and the half maximal inhibitory concentration of MELK inhibitor, OTS167, ranged from 9.3 to 60 nM. Additionally, OTS167 showed significant growth inhibitory effect against patient-derived ovarian cancer cells, regardless of their tumor locations, histologic subtypes and stages.

Conclusions: We demonstrated MELK as both a prognostic marker and a therapeutic target for ovarian cancer using clinical ovarian cancer samples. MELK inhibition by OTS167 may be an effective approach to treat ovarian cancer patients.
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http://dx.doi.org/10.3802/jgo.2020.31.e93DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593222PMC
November 2020

Ultradeep targeted sequencing of circulating tumor DNA in plasma of early and advanced breast cancer.

Cancer Sci 2021 Jan 4;112(1):454-464. Epub 2020 Nov 4.

Cancer Precision Medicine Center, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan.

We present a study to evaluate the feasibility and clinical utility of amplicon-based Oncomine Pan-Cancer cell-free assay to detect circulating tumor DNA (ctDNA) in patients with early or advanced breast cancer. In this study, 109 early and metastatic breast cancer patients were recruited before the initiation of treatment. ctDNA mutation profiles were assessed through unique molecular tagging (UMT) and ultradeep next generation sequencing (NGS). For patients with mutations, DNA from corresponding white blood cells (WBC) was sequenced to exclude variants of clonal-hematopoietic (CH) origin. UMT targeted sequencing from plasma of 109 patients achieved a median total coverage of 55 498X and a median molecular coverage of 4187X. Among 53 ctDNA positive samples, 38% were mutation positive by WBC sequencing, indicating potentially false-positive results contributed by CH origin. Prevalence of CH-related mutations was associated with age (P = 7.51 × 10 ). After exclusion of CH mutations, ctDNA detection rates were 37% for local or locally advanced breast cancer (stage I-III) and 81% for metastatic or recurrent breast cancer. The ctDNA detection rate correlated with disease stage (P = 2.60 × 10 ), nodal spread (P = 6.49 × 10 ) and the status of distant metastases (P = 5.00 × 10 ). ctDNA variants were detected mostly in TP53, PIK3CA and AKT1 genes, with variants showing therapeutic relevance. This pilot study endorses the use of targeted NGS for non-invasive molecular profiling of breast cancer. Paired sequencing of plasma ctDNA and WBC should be implemented to improve accurate interpretation of liquid biopsy.
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http://dx.doi.org/10.1111/cas.14697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780051PMC
January 2021

Phase II Adjuvant Cancer-specific Vaccine Therapy for Esophageal Cancer Patients Curatively Resected After Preoperative Therapy With Pathologically Positive Nodes; Possible Significance of Tumor Immune Microenvironment in its Clinical Effects.

Ann Surg 2020 Aug 26. Epub 2020 Aug 26.

*Department of Surgery, Kindai University Faculty of Medicine, Osaka, Japan †Department of Surgery, Ohita Nakamura Hospital, Ohita, Japan ‡Life Science Research Institute, Kindai University, Osaka, Japan §Clinical Research Center, Kindai University Hospital, Osaka, Japan ¶Division of Cell Biology, Biomedical Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan ||Division of Hospital Pathology, Kindai University Hospital, Osaka, Japan **Cancer Precision Medical Center, Tokyo, Japan.

Objectives: To elucidate the efficacy of adjuvant vaccine monotherapy using 3 Human Leukocyte Antigen (HLA)-A24-restricted tumor-specific peptide antigens for ESCC, upregulated lung cancer 10, cell division cycle associated 1, and KH domain-containing protein overexpressed in cancer 1.

Summary Of Background Data: ESCC patients with pathologically positive nodes (pN(+)) have a high risk for postoperative recurrence, despite curative resection after preoperative therapy. Subclinical micrometastases are an appropriate target for cancer vaccine.

Methods: This is a non-randomized prospective phase II clinical trial (UMIN000003557). ESCC patients curatively resected after preoperative therapy with pN(+) were allocated into the control and vaccine groups (CG and VG) according to the HLA-A status. One mg each of three epitope peptides was postoperatively injected 10 times weekly followed by 10 times biweekly to the VG. The primary and secondary endpoints were relapse-free survival (RFS) and esophageal cancer-specific survival (ECSS), respectively.

Results: Thirty were in the CG and 33 in the VG. No significant difference was observed in RFS between the CG and VG (5-year RFS: 32.5% vs 45.3%), but the recurrence rate significantly decreased with the number of peptides which induced antigen-specific cytotoxic T lymphocytes. The VG showed a significantly higher 5-year ECSS than the CG (60.0% vs 32.4%, P = 0.045) and this difference was more prominent in patients with CD8 and programmed death-ligand 1 double negative tumor (68.0% vs 17.7%, P = 0.010).

Conclusions: Our cancer peptide vaccine might improve the survival of ESCC patients, which is warranted to be verified in the phase III randomized controlled study.
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http://dx.doi.org/10.1097/SLA.0000000000003880DOI Listing
August 2020

Clinicopathologic significance of protein lysine methyltransferases in cancer.

Clin Epigenetics 2020 10 13;12(1):146. Epub 2020 Oct 13.

Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, 41 Medlars Drive, National Cancer Institute, Bethesda, MD, 20892, USA.

Protein lysine methyltransferases (PKMTs) constitute a large family of approximately 50 chromatin modifiers that mono-, di- and/or tri-methylate lysine residues on histone and non-histone substrates. With the advent of The Cancer Genome Atlas, it became apparent that this family of chromatin modifiers harbors frequent genetic and expression alterations in multiple types of cancer. In this regard, past and ongoing preclinical studies have provided insight into the mechanisms of action of some of these enzymes, laying the ground for the ongoing development of PKMT inhibitors as novel anticancer therapeutics. The purpose of this review is to summarize existing data obtained by different research groups through immunohistochemical analysis of the protein expression levels of PKMTs, and their respective clinicopathologic associations. We focused on studies that used immunohistochemistry to associate protein expression levels of specific PKMTs, as well as several established histone methylation marks, with clinicopathologic features and survival outcomes in various cancer types. We also review ongoing clinical trials of PKMT inhibitors in cancer treatment. This review underscores the clinical relevance and potential of targeting the family of PKMT enzymes as the next generation of cancer therapy.
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http://dx.doi.org/10.1186/s13148-020-00897-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7557092PMC
October 2020

Auto-antibody evaluation in idiopathic interstitial pneumonia and worse survival of patients with Ro52/TRIM21auto-antibody.

J Clin Biochem Nutr 2020 Sep 15;67(2):199-205. Epub 2020 May 15.

Department of Rheumatology, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, Japan.

Some patients with interstitial pneumonia (IP) have auto-antibodies, but do not fit the criteria for specific connective tissue diseases. Examination of auto-antibodies is recommended for diagnosis idiopathic pulmonary fibrosis. A prospective cohort study was performed in 285 patients with IP. Eleven auto-antibodies were assessed and patients were followed for 2 years. All 285 patients underwent the myositis panel test (MPT) for 11 auto-antibodies. Among them, 23.5% (67/285) of the patients had a positive MPT and 14.7% (42/285) had connective tissue diseases. Among the 49 MPT positive patients without connective tissue diseases, 29 patients (59.2%) were positive for Ro52, including 17 patients with Ro52 mono-positivity. Among interstitial pneumonia patients without connective tissue diseases, the Ro52 mono-positive patients showed worse at 2-years survival than those who were Ro52 negative ( = 0.022, HR = 5.88, 95% CI 1.29-26.75). Most of the Ro52 positive patients also showed a low titer of anti-nucleolar antibody. About 20% of IP patients had auto-antibodies detectable by the MPT, and Ro52 positive patients accounted for more than half of the MPT positive patients without connective tissue diseases. Detection of Ro52 auto-antibodies may be useful for assessing the risk of progression in idiopathic interstitial pneumonia patients without connective tissue diseases and a low anti-nucleolar antibody titer.
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http://dx.doi.org/10.3164/jcbn.20-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533866PMC
September 2020

Detection of circulating tumor DNA in patients of operative colorectal and gastric cancers.

Oncotarget 2020 Aug 25;11(34):3198-3207. Epub 2020 Aug 25.

Shin-Matsudo Chuo General Hospital, Matsudo, Japan.

Liquid biopsy is a non-invasive tool to examine the genetic profile of tumors by identification of mutated circulating tumor DNA (ctDNA), which is often analyzed by next generation sequencing (NGS) or droplet digital PCR (ddPCR) assay. We first examined the ctDNA mutation in pre-operative plasma samples obtained from 154 colorectal cancer (CRC) and 46 gastric cancer (GC) patients, using the NGS-based panel assay. The overall detection rate of mutated ctDNA was 72.0% (144 of 200 patients), and the panel-based screening identified 207 and 47 mutations from CRC and GC patients, respectively. The ddPCR analysis was then performed on post-operative samples of 77 patients, and detection of mutated ctDNA was earlier than imaging-based diagnosis in all of 6 patients who showed the tumor recurrences after surgery. Our data also revealed that patients with positive post-operation ctDNA level showed significant shorter recurrence-free survival compared to the patients with negative ctDNA level (HR 14.9; 95% CI, 0.7-313.5; < 0.0001). These findings suggested that screening of mutated ctDNA by liquid biopsy aids in identifying the patients at high risk of post-operative recurrence, and serial screening of ctDNA would allow to monitor the response after treatment and/or early detection of tumor recurrence.
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http://dx.doi.org/10.18632/oncotarget.27682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456613PMC
August 2020

Winter-spring temperature pattern is closely related to the onset of cambial reactivation in stems of the evergreen conifer Chamaecyparis pisifera.

Sci Rep 2020 08 31;10(1):14341. Epub 2020 Aug 31.

Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183-8509, Japan.

Temperature is an important factor for the cambial growth in temperate trees. We investigated the way daily temperatures patterns (maximum, average and minimum) from late winter to early spring affected the timing of cambial reactivation and xylem differentiation in stems of the conifer Chamaecyparis pisifera. When the daily temperatures started to increase earlier from late winter to early spring, cambial reactivation occurred earlier. Cambium became active when it achieves the desired accumulated temperature above the threshold (cambial reactivation index; CRI) of 13 °C in 11 days in 2013 whereas 18 days in 2014. This difference in duration required for achieving accumulated temperature can be explained with the variations in the daily temperature patterns in 2013 and 2014. Our formula for calculation of CRI predicted the cambial reactivation in 2015. A hypothetical increase of 1-4 °C to the actual daily maximum temperatures of 2013 and 2014 shifted the timing of cambial reactivation and had different effects on cambial reactivation in the two consecutive years because of variations in the actual daily temperatures patterns. Thus, the specific annual pattern of accumulation of temperature from late winter to early spring is a critical factor in determining the timing of cambial reactivation in trees.
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http://dx.doi.org/10.1038/s41598-020-70356-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458908PMC
August 2020

Enhancement of Migration and Invasion of Gastric Cancer Cells by IQGAP3.

Biomolecules 2020 08 17;10(8). Epub 2020 Aug 17.

Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan.

Although gastric cancer is one of the most common causes of cancer death in the world, mechanisms underlying this type of tumor have not been fully understood. In this study, we found that , a member of the gene family, was significantly up-regulated in human gastric cancer starting from the early stages of tumor progression. Overexpression of IQGAP3 in 293T and NIH3T3 cells, which have no endogenous IQGAP3 expression, resulted in morphological change with multiple dendritic-like protrusions and enhanced migration. Overexpression of IQGAP3 also led to reduced cell-cell adhesion in 293T cells, likely as a result of its interactions with e-cadherin or β-catenin proteins. Additionally, IQGAP3 accumulated along the leading edge of migrating cells and at the cleavage furrow of dividing cells. In contrast, suppression of IQGAP3 by short-interfering RNA (siRNA) markedly reduced invasion and anchorage-independent growth of MKN1 and TMK-1 gastric cancer cells. We further confirmed that IQGAP3 interacted with Rho family GTPases, and had an important role in cytokinesis. Taken together, we demonstrated that IQGAP3 plays critical roles in migration and invasion of human gastric cancer cells, and regulates cytoskeletal remodeling, cell migration and adhesion. These findings may open a new avenue for the diagnosis and treatment of gastric cancer.
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http://dx.doi.org/10.3390/biom10081194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465220PMC
August 2020

Clonal Hematopoiesis in Liquid Biopsy: From Biological Noise to Valuable Clinical Implications.

Cancers (Basel) 2020 Aug 14;12(8). Epub 2020 Aug 14.

Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan.

The use of blood liquid biopsy is being gradually incorporated into the clinical setting of cancer management. The minimally invasive nature of the usage of cell-free DNA (cfDNA) and its ability to capture the molecular alterations of tumors are great advantages for their clinical applications. However, somatic mosaicism in plasma remains an immense challenge for accurate interpretation of liquid biopsy results. Clonal hematopoiesis (CH) is part of the normal process of aging with the accumulation of somatic mutations and clonal expansion of hematopoietic stem cells. The detection of these non-tumor derived CH-mutations has been repeatedly reported as a source of biological background noise of blood liquid biopsy. Incorrect classification of CH mutations as tumor-derived mutations could lead to inappropriate therapeutic management. CH has also been associated with an increased risk of developing cardiovascular disease and hematological malignancies. Cancer patients, who are CH carriers, are more prone to develop therapy-related myeloid neoplasms after chemotherapy than non-carriers. The detection of CH mutations from plasma cfDNA analysis should be cautiously evaluated for their potential pathological relevance. Although CH mutations are currently considered as "false-positives" in cfDNA analysis, future studies should evaluate their clinical significance in healthy individuals and cancer patients.
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http://dx.doi.org/10.3390/cancers12082277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463455PMC
August 2020

Association of Neutrophil Extracellular Traps with the Development of Idiopathic Osteonecrosis of the Femoral Head.

Am J Pathol 2020 11 21;190(11):2282-2289. Epub 2020 Jul 21.

Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan. Electronic address:

Idiopathic osteonecrosis of the femoral head (ONFH) is defined as necrosis of osteocytes due to a non-traumatic ischemia of the femoral head. Iatrogenic glucocorticoid administration and habitual alcohol intake are regarded as risk factors. It has been suggested that glucocorticoid-induced activation of platelets contributes to the local blood flow disturbance of the femoral head. Both activated platelets and alcohol can induce neutrophil extracellular traps (NETs). To determine the association of NETs with the development of idiopathic ONFH, surgically resected femoral heads of patients with idiopathic ONFH and osteoarthritis were assessed for existence of NET-forming neutrophils by immunofluorescence staining. NET-forming neutrophils were present in small vessels surrounding the femoral head of patients with idiopathic ONFH but not osteoarthritis. Moreover, Wistar-Kyoto rats were intravenously injected with NET-forming neutrophils or neutrophils without NET induction, and then the ischemic state of the tissue around the femoral head was evaluated by immunohistochemistry for hypoxia-inducible factor-1α. NET-forming neutrophils circulated into the tissue around the femoral head, and hypoxia-inducible factor-1α expression in the tissue was higher compared with that of rats intravenously administered with neutrophils without NET induction. Furthermore, ischemic change of osteocytes was observed in the femoral head of rats given an i.v. injection of NET-forming neutrophils. The collective findings suggest that NETs are possibly associated with the development of idiopathic ONFH.
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http://dx.doi.org/10.1016/j.ajpath.2020.07.008DOI Listing
November 2020

SARS-CoV-2 genomic variations associated with mortality rate of COVID-19.

J Hum Genet 2020 Dec 22;65(12):1075-1082. Epub 2020 Jul 22.

Project for Immunogenomics, Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan.

The coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, has rapidly expanded to a global pandemic. However, numbers of infected cases, deaths, and mortality rates related to COVID-19 vary from country to country. Although many studies were conducted, the reasons of these differences have not been clarified. In this study, we comprehensively investigated 12,343 SARS-CoV-2 genome sequences isolated from patients/individuals in six geographic areas and identified a total of 1234 mutations by comparing with the reference SARS-CoV-2 sequence. Through a hierarchical clustering based on the mutant frequencies, we classified the 28 countries into three clusters showing different fatality rates of COVID-19. In correlation analyses, we identified that ORF1ab 4715L and S protein 614G variants, which are in a strong linkage disequilibrium, showed significant positive correlations with fatality rates (r = 0.41, P = 0.029 and r = 0.43, P = 0.022, respectively). We found that BCG-vaccination status significantly associated with the fatality rates as well as number of infected cases. In BCG-vaccinated countries, the frequency of the S 614G variant had a trend of association with the higher fatality rate. We also found that the frequency of several HLA alleles, including HLA-A*11:01, were significantly associated with the fatality rates, although these factors were associated with number of infected cases and not an independent factor to affect fatality rate in each country. Our findings suggest that SARS-CoV-2 mutations as well as BCG-vaccination status and a host genetic factor, HLA genotypes might affect the susceptibility to SARS-CoV-2 infection or severity of COVID-19.
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http://dx.doi.org/10.1038/s10038-020-0808-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375454PMC
December 2020

Intranodal Administration of Neoantigen Peptide-loaded Dendritic Cell Vaccine Elicits Epitope-specific T Cell Responses and Clinical Effects in a Patient with Chemorefractory Ovarian Cancer with Malignant Ascites.

Immunol Invest 2020 Jul 13:1-18. Epub 2020 Jul 13.

Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

Chemorefractory ovarian cancer has limited therapeutic options. Hence, new types of treatment including neoantigen-specific immunotherapy need to be investigated. Neoantigens represent promising targets for personalized cancer immunotherapy. We here describe the clinical and immunological effects of a neoantigen peptide-loaded DC-based immunotherapy in a patient with recurrent and chemoresistant ovarian cancer. A 71-year-old female patient with chemorefractory ovarian cancer and malignant ascites received intranodal vaccination of DCs loaded with four neoantigen peptides that were predicted by our immunogenomic pipeline. Following four rounds of vaccinations with this therapy, CA-125 levels were remarkably declined and tumor cells in the ascites were also decreased. Concordantly, the tumor-related symptoms such as respiratory discomfort improved without any adverse reactions. The reactivity against one HLA-A2402-restricted neoantigen peptide derived from a mutated PPM1 F protein was detected in lymphocytes from peripheral blood by IFN-γ ELISPOT assay. Furthermore, the neoantigen (PPM1 F mutant)-specific TCRs were detected in the tumor-infiltrating T lymphocytes post-vaccination. Our results showed that vaccination with intranodal injection of neoantigen peptide-loaded DCs may have clinical and immunological impacts on cancer treatment.
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http://dx.doi.org/10.1080/08820139.2020.1778721DOI Listing
July 2020

[A Case of Severe Aortic Thrombosis during the First Chemotherapy Regimen of CapeOX plus Bevacizumab for Metastatic Colon Cancer].

Gan To Kagaku Ryoho 2020 Jun;47(6):989-992

Dept. of Surgery, National Hospital Organization, Oita Medical Center.

A 61-year-old man underwent CapeOX plus bevacizumab chemotherapyafter right hemicolectomyfor metastatic ascending colon cancer. On the 7th dayafter the first administration, he had sudden abdominal pain and nausea. Contrast-enhanced computed tomographyrevealed aortic thrombosis and a superior mesenteric artery(SMA)embolism that was considered to be associated with bevacizumab. Bevacizumab was discontinued and anticoagulation therapyusing heparin and urokinase was performed. Brain infarction of the left middle cerebral arteryoccurred on the 15th dayafter the first administration and thrombectomywas performed. Anticoagulation therapyusing heparin, bayaspirin, and edoxaban tosilate hydrate was performed. The aortic thrombosis and SMA embolism resolved with treatment, but the patient died following an increase in peritoneal dissemination. It should be noted that unexpectedlysevere aortic thrombosis occurred during the first administration of CapeOX plus bevacizumab for metastatic colon cancer.
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June 2020

Plasma or Serum: Which Is Preferable for Mutation Detection in Liquid Biopsy?

Clin Chem 2020 07;66(7):946-957

Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

Background: Blood-based analysis of circulating tumor DNA (ctDNA) is a promising tool for cancer screening, monitoring relapse/recurrence and evaluating response to treatment. Although plasma is widely used to obtain ctDNA, biorepositories worldwide possess a huge number of serum samples and comparative studies on the use of serum vs plasma as ctDNA sources are essential.

Methods: We analyzed cell-free DNA (cfDNA) from matched EDTA-plasma and serum samples from healthy donors and patients with colorectal or lung cancer, and used targeted next-generation sequencing to evaluate mutation detection efficiency and reproducibility. Matched samples from healthy individuals were spiked with reference oligonucleotides and sequenced using the Ion-S5 Oncomine-Pan-Cancer panel. Detection efficiency in matched samples from patients with cancer was evaluated using 2 distinct gene panels and compared to mutations found in tissue-biopsy samples at diagnosis.

Results: Mean total cfDNA was 55% higher in serum samples and the presence of longer DNA fragments was significantly increased in serum compared with plasma samples (P = 0.0001 to 0.015). Spiked mutated nucleotides were detected in both samples, but allele frequencies (AF) were approximately half in serum compared with plasma, suggesting ctDNA from serum was more diluted by DNA of noncancerous origins. Matched samples from patients with cancer revealed that up to 44.8% of mutations with low AF were missed in serum samples and concordance rates with somatic mutations found in tissue biopsy at diagnosis was better in plasma samples.

Conclusion: The use of serum in retrospective studies should consider the limitations for detecting low AF mutations. Plasma is clearly preferable for prospective clinical applications of liquid biopsy.
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http://dx.doi.org/10.1093/clinchem/hvaa103DOI Listing
July 2020

Large-scale genome-wide association study in a Japanese population identifies novel susceptibility loci across different diseases.

Nat Genet 2020 07 8;52(7):669-679. Epub 2020 Jun 8.

Laboratory for Bone and Joint Diseases, RIKEN Center for Integrative Medical Sciences, Tokyo, Japan.

The overwhelming majority of participants in current genetic studies are of European ancestry. To elucidate disease biology in the East Asian population, we conducted a genome-wide association study (GWAS) with 212,453 Japanese individuals across 42 diseases. We detected 320 independent signals in 276 loci for 27 diseases, with 25 novel loci (P < 9.58 × 10). East Asian-specific missense variants were identified as candidate causal variants for three novel loci, and we successfully replicated two of them by analyzing independent Japanese cohorts; p.R220W of ATG16L2 (associated with coronary artery disease) and p.V326A of POT1 (associated with lung cancer). We further investigated enrichment of heritability within 2,868 annotations of genome-wide transcription factor occupancy, and identified 378 significant enrichments across nine diseases (false discovery rate < 0.05) (for example, NKX3-1 for prostate cancer). This large-scale GWAS in a Japanese population provides insights into the etiology of complex diseases and highlights the importance of performing GWAS in non-European populations.
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http://dx.doi.org/10.1038/s41588-020-0640-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968075PMC
July 2020

WHSC1 monomethylates histone H1 and induces stem-cell like features in squamous cell carcinoma of the head and neck.

Neoplasia 2020 08 16;22(8):283-293. Epub 2020 May 16.

Department of Medicine, University of Chicago, Chicago, USA; Department of Surgery, University of Chicago, Chicago, USA.

Squamous cell carcinoma of the head and neck (SCCHN) is a malignancy with poor outcomes, thus novel therapies are urgently needed. We recently showed that WHSC1 is necessary for the viability of SCCHN cells through H3K36 di-methylation. Here, we report the identification of its novel substrate, histone H1, and that WHSC1-mediated H1.4K85 mono-methylation may enhance stemness features in SCCHN cells. To identify proteins interacting with WHSC1 in SCCHN cells, WHSC1 immunoprecipitation and mass spectrometry identified H1 as a WHSC1-interacting candidate. In vitro methyltransferase assays showed that WHSC1 mono-methylates H1 at K85. We generated an H1K85 mono-methylation-specific antibody and confirmed that this methylation occurs in vivo. Sphere formation assays using SCC-35 cells stably expressing either wild-type (FLAG-H1.4-WT) or mutated (FLAG-H1.4K85A) vector with lysine 85 to alanine substitution which is not methylated, indicated a higher number of spheres in SCC-35 cells expressing the wild type than those with the mutant vector. SCC-35 cells expressing the wild type H1.4 proliferated faster than those expressing the mutated vector. RNA sequencing, RT-PCR and Western blotting of the FLAG-H1.4-WT or FLAG-H1.4K85A SCC-35 cells revealed that OCT4 levels were higher in wild type compared to mutant cells. These results were reproduced in SCC-35 cells genetically modified with CRISPR to express H1.4K85R. Chromatin immunoprecipitation showed that FLAG-H1.4K85A had decreased occupancy in the OCT4 gene compared to FLAG-H1.4-WT. This study supports that WHSC1 mono-methylates H1.4 at K85, it induces transcriptional activation of OCT4 and stemness features in SCCHN cells, providing rationale to target H1.4K85 mono-methylation through WHSC1 in SCCHN.
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http://dx.doi.org/10.1016/j.neo.2020.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265065PMC
August 2020

Imaging of lysophosphatidylcholine in an induced pluripotent stem cell-derived endothelial cell network.

Regen Ther 2020 Jun 18;14:299-305. Epub 2020 May 18.

Department of Biochemistry, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi, 321-0293, Japan.

Introduction: Vascular endothelial cell disorders are closely related to cardiovascular disease (CVD) and pulmonary diseases. Abnormal lipid metabolism in the endothelium leads to changes in cell signalling, and the expression of genes related to immunity and inflammation. It is therefore important to investigate the pathophysiology of vascular endothelial disorders in terms of lipid metabolism, using a disease model of endothelium.

Methods: Human induced pluripotent stem cell-derived endothelial cells (iECs) were cultured on a matrigel to form an iEC network. Lipids in the iEC network were investigated by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) analysis. Ion fragments obtained by mass spectrometry were analysed using an infusion method, involving precursor ion scanning with fragment ion.

Results: The MALDI TOF IMS analysis revealed co-localized intensity of peaks at / 592.1 and 593.1 in the iEC network. Tandem mass spectrometry (MS/MS) analysis by MALDI-imaging, in conjunction with precursor ion scanning using an infusion method with lipid extracts, identified that these precursor ions were lysophosphatidylcholine (LPC) (22:5) and its isotype.

Conclusion: The MALDI-imaging analysis showed that LPC (22:5) was abundant in an iEC network. As an in vitro test model for disease and potential therapy, present analysis methods using MALDI-imaging combined with, for example, mesenchymal stem cells (MSC) to a disease derived iEC network may be useful in revealing the changes in the amount and distribution of lipids under various stimuli.
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http://dx.doi.org/10.1016/j.reth.2020.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240204PMC
June 2020