Publications by authors named "Naoya Fujita"

155 Publications

Novel knock-in mouse model for the evaluation of the therapeutic efficacy and toxicity of human podoplanin-targeting agents.

Cancer Sci 2021 Mar 18. Epub 2021 Mar 18.

Division of Clinical Chemotherapy, The Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

Podoplanin is a key molecule for enhancing tumor-induced platelet aggregation. Podoplanin interacts with CLEC-2 on platelets via PLatelet Aggregation-inducing domains (PLAGs). Among our generated antibodies, those targeting the fourth PLAG domain (PLAG4) strongly suppress podoplanin-CLEC-2 binding and podoplanin-expressing tumor growth and metastasis. We previously performed a single-dose toxicity study of PLAG4-targeting anti-podoplanin-neutralizing antibodies and found no acute toxicity in cynomolgus monkeys. To confirm the therapeutic efficacy and toxicity of podoplanin-targeting antibodies, a syngeneic mouse model that enables repeated dose toxicity tests is needed. Replacement of mouse PLAG1-PLAG4 domains with human homologous domains drastically decreased the platelet-aggregating activity. Therefore, we searched the critical domain of the platelet-aggregating activity in mouse podoplanin and found that the mouse PLAG4 domain played a critical role in platelet aggregation, similar to the human PLAG4 domain. Human/mouse chimeric podoplanin, in which a limited region containing mouse PLAG4 was replaced with human homologous region, exhibited a similar platelet-aggregating activity to wild-type mouse podoplanin. Thus, we generated knock-in mice with human/mouse chimeric podoplanin expression (Pdpn mice). Our previously established PLAG4-targeting antibodies could suppress human/mouse chimeric podoplanin-mediated platelet aggregation and tumor growth in Pdpn mice. Repeated treatment of Pdpn mice with antibody-dependent cell-mediated cytotoxicity activity-possessing PG4D2 antibody did not result in toxicity or changes in hematological and biochemical parameters. Our results suggest that anti-podoplanin-neutralizing antibodies could be used safely as novel anti-tumor agents. Our generated Pdpn mice are useful for investigating the efficacy and toxicity of human podoplanin-targeting drugs.
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http://dx.doi.org/10.1111/cas.14891DOI Listing
March 2021

Monitoring EGFR C797S mutation in Japanese NSCLC patients with serial cell free DNA evaluation using digital droplet PCR.

Cancer Sci 2021 Mar 8. Epub 2021 Mar 8.

Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31, Ariake, Koto-ku, Tokyo, 135-8550, Japan.

Osimertinib is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) effective to both treatment naïve and T790M mutated EGFR-TKI resistant NSCLC patients. As the reported EGFR C797S mutation is the major osimertinib resistance mechanism. This study aimed to monitor the EGFR C797S mutation along the osimertinib treatment in Japanese patients using a droplet digital PCR (ddPCR). In the first cohort, C797S detection was validated with tumor specimens and/or plasma samples from 26 patients using ddPCR with custom designed probe detecting and discriminating T790M and C797S in cis and trans positions. In the second cohort, 18 patients detected EGFR-T790M who were going to start osimertinib were analyzed using ddPCR by collecting the plasma samples every month from the beginning of osimertinib. In the first cohort, C797S was detected in 15.4% of patients and C797S and T790M in cis and trans position was distinguished using ddPCR. In the second cohort, serial cfDNA evaluation revealed that the rate of EGFR mutation changes with disease state and detected increases of EGFR mutation including C797S several months before the diagnosis of disease progression. As same as first cohort, C797S and T790M in cis and trans position was distinguished by ddPCR at disease progression. Coincidentally, in the first cohort next generation sequencing detected NRAS Q61K mutation and the resistance with NRAS Q61K mutation was overcome by trametinib. In the second cohort, serial cfDNA analysis was useful to evaluate bone oligo-progression and local radiation therapy to it.
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http://dx.doi.org/10.1111/cas.14879DOI Listing
March 2021

Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer.

Nat Commun 2021 02 24;12(1):1261. Epub 2021 Feb 24.

Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

ALK gene rearrangement was observed in 3%-5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI-resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.
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http://dx.doi.org/10.1038/s41467-021-21396-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904790PMC
February 2021

TAS-119, a novel selective Aurora A and TRK inhibitor, exhibits antitumor efficacy in preclinical models with deregulated activation of the Myc, β-Catenin, and TRK pathways.

Invest New Drugs 2021 Jan 6. Epub 2021 Jan 6.

Discovery and Preclinical Research Division, Taiho Pharmaceutical Co., Ltd, 3 Okubo, Tsukuba, Ibaraki, 300-2611, Japan.

Aurora kinase A, a mitotic kinase that is overexpressed in various cancers, is a promising cancer drug target. Here, we performed preclinical characterization of TAS-119, a novel, orally active, and highly selective inhibitor of Aurora A. TAS-119 showed strong inhibitory effect against Aurora A, with an IC value of 1.04 nmol/L. The compound was highly selective for Aurora A compared with 301 other protein kinases, including Aurora kinase B. TAS-119 induced the inhibition of Aurora A and accumulation of mitotic cells in vitro and in vivo. It suppressed the growth of various cancer cell lines harboring MYC family amplification and CTNNB1 mutation in vitro. In a xenograft model of human lung cancer cells harboring MYC amplification and CTNNB1 mutation, TAS-119 showed a strong antitumor activity at well-tolerated doses. TAS-119 induced N-Myc degradation and inhibited downstream transcriptional targets in MYCN-amplified neuroblastoma cell lines. It also demonstrated inhibitory effect against tropomyosin receptor kinase (TRK)A, TRKB, and TRKC, with an IC value of 1.46, 1.53, and 1.47 nmol/L, respectively. TAS-119 inhibited TRK-fusion protein activity and exhibited robust growth inhibition of tumor cells via a deregulated TRK pathway in vitro and in vivo. Our study indicates the potential of TAS-119 as an anticancer drug, especially for patients harboring MYC amplification, CTNNB1 mutation, and NTRK fusion.
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http://dx.doi.org/10.1007/s10637-020-01019-9DOI Listing
January 2021

Perinatal factors contributing to chronic kidney disease in a cohort of Japanese children with very low birth weight.

Pediatr Nephrol 2021 Apr 17;36(4):953-960. Epub 2020 Oct 17.

Department of Pediatric Nephrology, Tokyo Metropolitan Children's Medical Center, Tokyo, Japan.

Background: Developmental programming of chronic kidney disease (CKD) in young adults is linked to preterm birth and intrauterine growth restriction (IUGR). Which confers a higher risk of progression to chronic kidney damage in children with very low birth weight (VLBW; born weighing < 1500 g): prematurity or IUGR?

Methods: This is a national historical cohort study of children with VLBW cared for in perinatal medical centers in Japan. Predictive factors included three latent variables (prematurity, IUGR, stress during neonatal period) and eight observed variables (gestational age, birth weight Z-score, maternal age, duration of treatment with antibiotics and diuretics, maternal smoking, late-onset circulatory collapse, kidney dysfunction) during the perinatal period. The primary endpoint was estimated glomerular filtration rate (eGFR) at age ≥ 3 years. A structural equation model was used to examine the pathologic constitution.

Results: The 446 children with VLBW included 253 boys and 193 girls, of mean age 5.8 ± 2.6 years and mean eGFR 111.7 ml/min/1.73 m at last encounter. Pathway analyses showed intrauterine malnutrition (β = 0.85) contributed more to chronic kidney damage than stress during the neonatal period (β = - 0.19) and prematurity (β = 0.12), and kidney dysfunction and late-onset circulatory collapse were important observed variables in stress during the neonatal period.

Conclusions: IUGR was more harmful to future kidneys of VLBW neonates. Neonatal kidney dysfunction and late-onset circulatory collapse were important risk factors for subsequent CKD development. This emphasizes the need for obstetricians to monitor for fetal growth restriction and neonatologists to minimize neonatal stress to prevent CKD in later life.
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http://dx.doi.org/10.1007/s00467-020-04791-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910374PMC
April 2021

The long-acting C5 inhibitor, ravulizumab, is efficacious and safe in pediatric patients with atypical hemolytic uremic syndrome previously treated with eculizumab.

Pediatr Nephrol 2021 Apr 13;36(4):889-898. Epub 2020 Oct 13.

Division of Pediatric Nephrology, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA, USA.

Background: Atypical hemolytic uremic syndrome (aHUS) is a rare, complement-mediated disease associated with poor outcomes if untreated. Ravulizumab, a long-acting C5 inhibitor developed through minimal, targeted modifications to eculizumab was recently approved for the treatment of aHUS. Here, we report outcomes from a pediatric patient cohort from the ravulizumab clinical trial (NCT03131219) who were switched from chronic eculizumab to ravulizumab treatment.

Methods: Ten patients received a loading dose of ravulizumab on Day 1, followed by maintenance doses administered initially on Day 15, and then, every 4-8 weeks thereafter, depending on body weight. All patients completed the initial evaluation period of 26 weeks and entered the extension period.

Results: No patients required dialysis at any point throughout the study. The median estimated glomerular filtration rate values remained stable during the trial: 99.8 mL/min/1.73m at baseline, 93.5 mL/min/1.73m at 26 weeks, and 104 mL/min/1.73m at 52 weeks. At last available follow-up, all patients were in the same chronic kidney disease stage as recorded at baseline. Hematologic variables (platelets, lactate dehydrogenase, and hemoglobin) also remained stable throughout the initial evaluation period and up to the last available follow-up. All patients experienced adverse events; the most common were upper respiratory tract infection (40%) and oropharyngeal pain (30%). There were no meningococcal infections reported, no deaths occurred, and no patients discontinued during the study.

Conclusions: Overall, treatment with ravulizumab in pediatric patients with aHUS who were previously treated with eculizumab resulted in stable kidney and hematologic parameters, with no unexpected safety concerns when administered every 4-8 weeks.

Trial Registration: Trial identifiers: Trial ID: ALXN1210-aHUS-312 Clinical trials.gov : NCT03131219 EudraCT number: 2016-002499-29 Graphical abstract.
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http://dx.doi.org/10.1007/s00467-020-04774-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910247PMC
April 2021

Cryo-EM structures reveal distinct mechanisms of inhibition of the human multidrug transporter ABCB1.

Proc Natl Acad Sci U S A 2020 10 5;117(42):26245-26253. Epub 2020 Oct 5.

Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland;

ABCB1 detoxifies cells by exporting diverse xenobiotic compounds, thereby limiting drug disposition and contributing to multidrug resistance in cancer cells. Multiple small-molecule inhibitors and inhibitory antibodies have been developed for therapeutic applications, but the structural basis of their activity is insufficiently understood. We determined cryo-EM structures of nanodisc-reconstituted, human ABCB1 in complex with the Fab fragment of the inhibitory, monoclonal antibody MRK16 and bound to a substrate (the antitumor drug vincristine) or to the potent inhibitors elacridar, tariquidar, or zosuquidar. We found that inhibitors bound in pairs, with one molecule lodged in the central drug-binding pocket and a second extending into a phenylalanine-rich cavity that we termed the "access tunnel." This finding explains how inhibitors can act as substrates at low concentration, but interfere with the early steps of the peristaltic extrusion mechanism at higher concentration. Our structural data will also help the development of more potent and selective ABCB1 inhibitors.
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http://dx.doi.org/10.1073/pnas.2010264117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7585025PMC
October 2020

Efficacy of EGFR tyrosine kinase inhibitors in patients having EGFR-activating mutations with or without BIM polymorphisms.

Cancer Chemother Pharmacol 2020 Oct 18;86(4):517-525. Epub 2020 Sep 18.

Department of Thoracic Medical Oncology, The Cancer Institute Hospital, Japanese Foundation for Cancer Research, 3-8-31, Ariake, Koto-ku, Tokyo, 135-8550, Japan.

Purpose: Patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer with BIM deletion polymorphism may have a limited response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, some results of previous reports are discordant. It is necessary to evaluate the relationship between BIM polymorphism and the efficacy of EGFR-TKIs.

Methods: We retrospectively analyzed patients treated with EGFR-TKIs. We collected serum samples from patients before EGFR-TKI administration. We analyzed BIM deletion polymorphism and BIM single nucleotide polymorphism in exon 5 c465C > T by the Invader assay.

Results: BIM deletion polymorphism was identified in 27 of 194 patients (13.9%). BIM single nucleotide polymorphism was identified in 29 of 194 patients (14.9%). The overall response ratio was 81.5% in patients with BIM deletion polymorphism, 89.7% with BIM single nucleotide polymorphism, and 83.6% with BIM wild type. Median progression-free survival was 10.3 months with BIM deletion polymorphism, 8.5 months with BIM single nucleotide polymorphism, and 10.4 months with BIM wild type. Overall survival was 38.4 months with BIM deletion polymorphism, 29.1 months with BIM single nucleotide polymorphism, and 31.6 months with BIM wild type. There were no significant differences between the groups in overall response ratio, progression-free survival, and overall survival.

Conclusions: BIM polymorphism does not affect EGFR-TKI efficacy.
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http://dx.doi.org/10.1007/s00280-020-04136-7DOI Listing
October 2020

Management of a Preterm Infant with Renal Tubular Dysgenesis: A Case Report and Review of the Literature.

Tohoku J Exp Med 2020 09;252(1):9-14

Department of Pediatrics, Fujita Health University School of Medicine.

Renal tubular dysgenesis (RTD) is the absence or poor development of the renal proximal tubules caused by gene mutations in the renin-angiotensin system. Although RTD has been considered fatal, improving neonatal intensive care management has enhanced survival outcomes. However, little has been reported on the survival of extremely preterm infants. This study reports the survival of an extremely preterm infant with RTD and discusses the appropriate management of RTD by reviewing the literature. A female infant weighing 953 g was delivered at 27 weeks' gestation by Cesarean section because of oligohydramnios. She exhibited severe persistent pulmonary hypertension, severe systemic hypotension, and renal dysfunction shortly after birth. Respiratory management was successfully undertaken using nitric oxide inhalation and high-frequency oscillatory ventilation. Desmopressin was effective in maintaining her blood pressure and urinary output. She was diagnosed with RTD based on genetic testing, which revealed a compound heterozygous mutation in the angiotensin-converting enzyme gene in exon 18 (c.2689delC; p.Pro897fs) and exon 20 (c.3095dupT; p.Leu1032fs). At 2 years, she started receiving oral fludrocortisone for treating persistently high serum creatinine levels, which was attributed to nephrogenic diabetes insipidus caused by RTD. Subsequently, her urine output decreased, and renal function was successfully maintained. Currently, there is no established treatment for RTD. Considering cases reported to date, treatment with vasopressin and fludrocortisone appears to be most effective for survival and maintenance of renal function in patients with RTD. This study presents the successful management of RTD using this strategy in an extremely preterm infant.
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http://dx.doi.org/10.1620/tjem.252.9DOI Listing
September 2020

Diabetes-associated Necrotizing Fasciitis of the Foot.

Intern Med 2020 11 14;59(21):2805-2806. Epub 2020 Jul 14.

Department of General Medicine, National Defense Medical College, Japan.

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http://dx.doi.org/10.2169/internalmedicine.5001-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691028PMC
November 2020

IgG4-related Paravertebral Mass and Peri-aortitis.

Intern Med 2020 10 30;59(19):2447-2448. Epub 2020 Jun 30.

Department of General Medicine, National Defense Medical College, Japan.

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http://dx.doi.org/10.2169/internalmedicine.4770-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644484PMC
October 2020

Effect of Exclusive Enteral Nutrition on Renal Function for Granulomatous Interstitial Nephritis Associated With Crohn Disease.

Inflamm Bowel Dis 2020 Oct;26(11):e142-e143

Department of Infection and Immunology, Aichi Children's Health and Medical Center, Aichi, Japan.

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http://dx.doi.org/10.1093/ibd/izaa164DOI Listing
October 2020

Recent Topics Concerning Combined Hepatocellular Cholangiocarcinoma.

Kurume Med J 2020 Jul 1;66(1):29-36. Epub 2020 May 1.

Department of Pathology, Kurume University School of Medicine.

Combined hepatocellular-cholangiocarcinoma (CHC) is a relatively rare tumor with an incidence range of 1.0-4.7%. CHC is defined as a tumor containing unequivocal, intimately mixed components of both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. The recent development of biochemical methodologies and cancer stem cell theory have paved the way for a clearer understanding of the histogenesis of CHC. The latest edited WHO classification published in 2010 adopted the concept of stem cell/hepatic progenitor cells in the pathological classification of CHC. Although this classification includes novel and unique concepts of histogenesis and facilitates the recognition of CHC, there are several problems with it in practice. To reduce confusion, an international group of hepatic pathologists, radiologists, surgeons, and clinicians formulated a nomenclature for CHC and issued a consensus article in 2018. In this review article, we discuss the problems with the latest WHO classification and introduce recent topics concerning CHC from pathologic and genetic points of view.
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http://dx.doi.org/10.2739/kurumemedj.MS661014DOI Listing
July 2020

Prophylactic amoxicillin for the prevention of meningococcal infection in infants with atypical hemolytic uremic syndrome under treatment with eculizumab: a report of two cases.

CEN Case Rep 2020 08 2;9(3):247-251. Epub 2020 Apr 2.

Department of Nephrology, Aichi Children's Health and Medical Center, 7-426 Morioka-cho, Obu, Aichi, 474-8710, Japan.

Eculizumab, a humanized monoclonal antibody to complement C5, is a therapeutic drug for atypical hemolytic-uremic syndrome (aHUS) that inhibits the terminal pathway of complement. Patients on eculizumab therapy may become more susceptible to infection with capsule-forming bacteria, including meningococci. Therefore, meningococcal vaccination is required for patients who are on eculizumab therapy. However, the means to prevent meningococcal infection in infants who cannot be vaccinated with the available meningococcal vaccine have not yet been established internationally. In two infants with aHUS at 4-5 months after birth, prophylactic oral amoxicillin was administered, and meningococcal infection was not detected during the period between the initiation of eculizumab therapy and the administration of meningococcal vaccine. Neither adverse events related to amoxicillin nor thrombotic microangiopathy occurred during the treatment. Thus, oral administration of amoxicillin may be effective for preventing meningococcal infection under treatment with eculizumab in infants who have not received meningococcal vaccination.
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http://dx.doi.org/10.1007/s13730-020-00465-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7320125PMC
August 2020

Primary Cutaneous Diffuse Large B-cell Lymphoma, Leg Type.

Intern Med 2020 07 2;59(14):1785. Epub 2020 Apr 2.

Department of General Medicine, National Defense Medical College, Japan.

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http://dx.doi.org/10.2169/internalmedicine.4497-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434555PMC
July 2020

Medullary Cystic Kidney Disease and Focal Segmental Glomerulosclerosis Caused by a Compound Heterozygous Mutation in TTC21B.

Intern Med 2020 Jul 2;59(14):1735-1738. Epub 2020 Apr 2.

Pediatric Nephrology, Aichi Children's Health & Medical Center, Japan.

Mutations in the TTC21B gene have been identified in patients with nephronophthisis and were recently found in some patients with focal segmental glomerulosclerosis. We herein report a Japanese boy with end-stage renal disease due to medullary polycystic kidney disease and primary focal segmental glomerulosclerosis. Next-generation sequencing detected a new compound heterozygous missense mutation in the TTC21B gene. His renal pathological findings and gene mutations have not been previously reported in patients with ciliopathy. For children with severe renal dysfunction, mutations in the TTC21B gene cause both ciliopathy characterized by bilateral polycystic kidney disease and primary focal segmental glomerulosclerosis.
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http://dx.doi.org/10.2169/internalmedicine.4266-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434538PMC
July 2020

Kidney function of Japanese children undergoing kidney transplant with preemptive therapy for cytomegalovirus infection.

Transpl Infect Dis 2020 Jun 11;22(3):e13271. Epub 2020 Mar 11.

Ichinomiya Medical Treatment and Habilitation Center, Aichi, Japan.

Background: Cytomegalovirus (CMV) infection is one of the major factors that affect morbidity and mortality in kidney transplant (KTx) patients. The rate of CMV seropositivity in children before KTx is lower than that in adults; therefore, pediatric KTx patients have a higher risk of CMV infection. In Japanese pediatric KTx patients, preemptive therapy for CMV infection is a main conventional therapy. This study investigated whether this preemptive treatment would affect kidney function at 2 years post-KTx.

Methods: A total of 163 patients, that is approximately half of the Japanese pediatric KTx patients nationwide, were recruited to participate in our study. We compared the values of the sequential estimated glomerular filtration rate (eGFR) at two years post-KTx and other influencing factors in CMV viremia, CMV disease, and no-infection groups.

Results: Cytomegalovirus infection after KTx occurred in 75 patients (46.0%), 38.7% of whom developed CMV disease. The sequential eGFR values post-KTx did not differ significantly between the three groups. CMV infection was not significantly correlated with other factors, other infections (including Epstein-Barr [EB] virus infection), acute rejection (AR), or adverse events. Only prolonged duration of total hospitalization was significantly associated with CMV infection (P = .002). In the multivariate analysis, younger age, CMV infection, and adverse effects were independently significantly related to prolonged total hospitalization.

Conclusion: Preemptive therapy for CMV infection evidenced by viremia and disease did not significantly influence kidney function in Japanese pediatric KTx patients at two years after the operation.
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http://dx.doi.org/10.1111/tid.13271DOI Listing
June 2020

Drug resistance mechanisms in Japanese anaplastic lymphoma kinase-positive non-small cell lung cancer and the clinical responses based on the resistant mechanisms.

Cancer Sci 2020 Mar 8;111(3):932-939. Epub 2020 Feb 8.

Department of Thoracic Medical Oncology, The Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan.

The treatment for anaplastic lymphoma kinase (ALK)-positive lung cancer has been rapidly evolving since the introduction of several ALK tyrosine kinase inhibitors (ALK-TKI) in clinical practice. However, the acquired resistance to these drugs has become an important issue. In this study, we collected a total of 112 serial biopsy samples from 32 patients with ALK-positive lung cancer during multiple ALK-TKI treatments to reveal the resistance mechanisms to ALK-TKI. Among 32 patients, 24 patients received more than two ALK-TKI. Secondary mutations were observed in 8 of 12 specimens after crizotinib failure (G1202R, G1269A, I1171T, L1196M, C1156Y and F1245V). After alectinib failure, G1202R and I1171N mutations were detected in 7 of 15 specimens. G1202R, F1174V and G1202R, and P-gp overexpression were observed in 3 of 7 samples after ceritinib treatment. L1196M + G1202R, a compound mutation, was detected in 1 specimen after lorlatinib treatment. ALK-TKI treatment duration was longer in the on-target treatment group than that in the off-target group (13.0 vs 1.2 months). In conclusion, resistance to ALK-TKI based on secondary mutation in this study was similar to that in previous reports, except for crizotinib resistance. Understanding the appropriate treatment matching resistance mechanisms contributes to the efficacy of multiple ALK-TKI treatment strategies.
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http://dx.doi.org/10.1111/cas.14314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060465PMC
March 2020

Overcoming resistance by ALK compound mutation (I1171S + G1269A) after sequential treatment of multiple ALK inhibitors in non-small cell lung cancer.

Thorac Cancer 2020 03 13;11(3):581-587. Epub 2020 Jan 13.

Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

Background: Anaplastic lymphoma kinase (ALK) fusion genes are found in 3%-5% of non-small cell lung cancers (NSCLCs). ALK inhibitors show a very high response rate to ALK-positive NSCLCs. However, the emergence of acquired resistance is inevitable. In this study, we investigated the drugs for overcoming resistance especially compound mutations after sequential treatment with crizotinib, alectinib, and lorlatinib.

Method: Next-generation sequencing (NGS) and Sanger sequencing were performed on a liver biopsy tissue obtained from a clinical case. Ba/F3 cells in which mutant EML4-ALK were overexpressed were prepared, and cell viability assay and immunoblotting were performed to check the sensitivity of five independent ALK inhibitors.

Results: I1171S + G1269A double mutation was identified by NGS and Sanger sequencing on a liver biopsy tissue from a patient who relapsed on lorlatinib treatment. Ceritinib and brigatinib-but not other ALK inhibitors-were active against the compound mutations in the cell line model.

Conclusions: With the sequential ALK inhibitors treatment, cancer cells accumulate new mutations in addition to mutations acquired previously. The identified compound mutation (I1171S + G1269A) was found to be sensitive to ceritinib and brigatinib, and indeed the patient's tumor partially responded to ceritinib.

Key Points: ALK compound mutation was found in a clinical sample that was resistant to lorlatinib after sequential ALK-tyrosine kinase inhibitor (TKI) treatment. Ceritinib and brigatinib are potential overcoming drugs against ALK I1171S + G1269A double mutation.
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http://dx.doi.org/10.1111/1759-7714.13299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049522PMC
March 2020

The new-generation selective ROS1/NTRK inhibitor DS-6051b overcomes crizotinib resistant ROS1-G2032R mutation in preclinical models.

Nat Commun 2019 08 9;10(1):3604. Epub 2019 Aug 9.

Daiichi Sankyo Co., Ltd, Tokyo, 140-8710, Japan.

ROS1 gene rearrangement was observed in around 1-2 % of NSCLC patients and in several other cancers such as cholangiocarcinoma, glioblastoma, or colorectal cancer. Crizotinib, an ALK/ROS1/MET inhibitor, is highly effective against ROS1-rearranged lung cancer and is used in clinic. However, crizotinib resistance is an emerging issue, and several resistance mechanisms, such as secondary kinase-domain mutations (e.g., ROS1-G2032R) have been identified in crizotinib-refractory patients. Here we characterize a new selective ROS1/NTRK inhibitor, DS-6051b, in preclinical models of ROS1- or NTRK-rearranged cancers. DS-6051b induces dramatic growth inhibition of both wild type and G2032R mutant ROS1-rearranged cancers or NTRK-rearranged cancers in vitro and in vivo. Here we report that DS-6051b is effective in treating ROS1- or NTRK-rearranged cancer in preclinical models, including crizotinib-resistant ROS1 positive cancer with secondary kinase domain mutations especially G2032R mutation which is highly resistant to crizotinib as well as lorlatinib and entrectinib, next generation ROS1 inhibitors.
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http://dx.doi.org/10.1038/s41467-019-11496-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688997PMC
August 2019

TAS4464, A Highly Potent and Selective Inhibitor of NEDD8-Activating Enzyme, Suppresses Neddylation and Shows Antitumor Activity in Diverse Cancer Models.

Mol Cancer Ther 2019 07 15;18(7):1205-1216. Epub 2019 May 15.

Discovery and Preclinical Research Division, Taiho Pharmaceutical Co., Ltd, Tsukuba, Japan.

NEDD8-activating enzyme (NAE) is an essential E1 enzyme of the NEDD8 conjugation (neddylation) pathway, which controls cancer cell growth and survival through activation of cullin-RING ubiquitin ligase complexes (CRL). In this study, we describe the preclinical profile of a novel, highly potent, and selective NAE inhibitor, TAS4464. TAS4464 selectively inhibited NAE relative to the other E1s UAE and SAE. TAS4464 treatment inhibited cullin neddylation and subsequently induced the accumulation of CRL substrates such as CDT1, p27, and phosphorylated IκBα in human cancer cell lines. TAS4464 showed greater inhibitory effects than those of the known NAE inhibitor MLN4924 both in enzyme assay and in cells. Cytotoxicity profiling revealed that TAS4464 is highly potent with widespread antiproliferative activity not only for cancer cell lines, but also patient-derived tumor cells. TAS4464 showed prolonged target inhibition in human tumor xenograft mouse models; weekly or twice a week TAS4464 administration led to prominent antitumor activity in multiple human tumor xenograft mouse models including both hematologic and solid tumors without marked weight loss. As a conclusion, TAS4464 is the most potent and highly selective NAE inhibitor reported to date, showing superior antitumor activity with prolonged target inhibition. It is, therefore, a promising agent for the treatment of a variety of tumors including both hematologic and solid tumors. These results support the clinical evaluation of TAS4464 in hematologic and solid tumors.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0644DOI Listing
July 2019

Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non-small cell lung cancer.

J Exp Med 2019 04 14;216(4):982-1000. Epub 2019 Mar 14.

Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan

Immune checkpoint blockade against programmed cell death 1 (PD-1) and its ligand PD-L1 often induces durable tumor responses in various cancers, including non-small cell lung cancer (NSCLC). However, therapeutic resistance is increasingly observed, and the mechanisms underlying anti-PD-L1 (aPD-L1) antibody treatment have not been clarified yet. Here, we identified two unique secreted PD-L1 splicing variants, which lacked the transmembrane domain, from aPD-L1-resistant NSCLC patients. These secreted PD-L1 variants worked as "decoys" of aPD-L1 antibody in the HLA-matched coculture system of iPSC-derived CD8 T cells and cancer cells. Importantly, mixing only 1% MC38 cells with secreted PD-L1 variants and 99% of cells that expressed wild-type PD-L1 induced resistance to PD-L1 blockade in the MC38 syngeneic xenograft model. Moreover, anti-PD-1 (aPD-1) antibody treatment overcame the resistance mediated by the secreted PD-L1 variants. Collectively, our results elucidated a novel resistant mechanism of PD-L1 blockade antibody mediated by secreted PD-L1 variants.
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http://dx.doi.org/10.1084/jem.20180870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446862PMC
April 2019

Prediction of ALK mutations mediating ALK-TKIs resistance and drug re-purposing to overcome the resistance.

EBioMedicine 2019 Mar 17;41:105-119. Epub 2019 Jan 17.

Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan. Electronic address:

Background: Alectinib has shown a greater efficacy to ALK-rearranged non-small-cell lung cancers in first-line setting; however, most patients relapse due to acquired resistance, such as secondary mutations in ALK including I1171N and G1202R. Although ceritinib or lorlatinib was shown to be effective to these resistant mutants, further resistance often emerges due to ALK-compound mutations in relapse patients following the use of ceritinib or lorlatinib. However, the drug for overcoming resistance has not been established yet.

Methods: We established lorlatinib-resistant cells harboring ALK-I1171N or -G1202R compound mutations by performing ENU mutagenesis screening or using an in vivo mouse model. We performed drug screening to overcome the lorlatinib-resistant ALK-compound mutations. To evaluate these resistances in silico, we developed a modified computational molecular dynamic simulation (MP-CAFEE).

Findings: We identified 14 lorlatinib-resistant ALK-compound mutants, including several mutants that were recently discovered in lorlatinib-resistant patients. Some of these compound mutants were found to be sensitive to early generation ALK-TKIs and several BCR-ABL inhibitors. Using our original computational simulation, we succeeded in demonstrating a clear linear correlation between binding free energy and in vitro experimental IC value of several ALK-TKIs to single- or compound-mutated EML4-ALK expressing Ba/F3 cells and in recapitulating the tendency of the binding affinity reduction by double mutations found in this study. Computational simulation revealed that ALK-L1256F single mutant conferred resistance to lorlatinib but increased the sensitivity to alectinib.

Interpretation: We discovered lorlatinib-resistant multiple ALK-compound mutations and an L1256F single mutation as well as the potential therapeutic strategies for these ALK mutations. Our original computational simulation to calculate the binding affinity may be applicable for predicting resistant mutations and for overcoming drug resistance in silico. FUND: This work was mainly supported by MEXT/JSPS KAKENHI Grants and AMED Grants.
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http://dx.doi.org/10.1016/j.ebiom.2019.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441848PMC
March 2019

A safety study of newly generated anti-podoplanin-neutralizing antibody in cynomolgus monkey ().

Oncotarget 2018 Sep 7;9(70):33322-33336. Epub 2018 Sep 7.

The Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

Hematogenous metastases are enhanced by platelet aggregation induced by tumor cell-platelet interaction. Podoplanin is a key molecule to enhance the platelet aggregation and interacts with C-type lectin-like receptor 2 (CLEC-2) on platelet PLAG domains. Our previous reports have shown that blocking podoplanin binding to platelets by neutralizing antibody specific to PLAG4 domain strongly reduces hematogenous metastasis. However, podoplanin is expressed in a variety of normal tissues such as lymphatic vessels and the question remains whether treatment of tumors with anti-podoplanin neutralizing antibodies would be toxic. Monkeys are the most suitable species for that purpose. PLAG3 and PLAG4 domains had high homology among various monkey species and human. PLAG domain deleted mutants were indicated that monkey PLAG4 domain played a more crucial role in podoplanin-induced platelet aggregation than did the PLAG3 domain as in human. Moreover, newly established neutralizing antibodies (1F6, 2F7, and 3F4) targeting the monkey PLAG4 domain blocked interaction between monkey podoplanin and CLEC-2. Especially, the 2F7 neutralizing antibody strongly suppressed platelet aggregation and pulmonary metastasis. Furthermore, inhibiting podoplanin function with 2F7 neutralizing antibody exhibited no acute toxicity in cynomolgus monkeys. Our results suggested that targeting podoplanin with specific neutralizing antibodies may be an effective anticancer treatment.
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http://dx.doi.org/10.18632/oncotarget.26055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161800PMC
September 2018

Solute Clearance and Fluid Removal: Large-Dose Cyclic Tidal Peritoneal Dialysis.

Ther Apher Dial 2019 Apr 24;23(2):180-186. Epub 2018 Oct 24.

Pediatric Nephrology, Aichi Children's Health & Medical Center, Aichi, Japan.

Large-dose cyclic tidal peritoneal dialysis (TPD) is an original prescription of TPD involving frequent infusion and drainage of the dialysate to increase weekly urea clearance normalized to total body water (Kt/V ) and fluid removal. This study aimed to evaluate the efficiency of solute clearance and fluid removal achieved with large-dose cyclic TPD compared to that achieved with nightly peritoneal dialysis (NPD). Seventeen patients with end-stage renal disease, for whom maintenance PD was changed from NPD to large-dose cyclic TPD, were enrolled. Their median age at administration of PD was 4.9 years. Kt/V and fluid removal were compared between large-dose cyclic TPD and NPD. The median peritoneal Kt/V achieved with NPD and large-dose cyclic TPD was 1.5 and 2.7, respectively. The median peritoneal Kt/V per hour with large-dose cyclic TPD was significantly higher than that with NPD (P = 0.0003). Among nine patients who used dialysates with the same glucose concentration for both NPD and large-dose cyclic TPD, nightly fluid removal amount per hour with large-dose cyclic TPD was significantly higher than that with NPD (P = 0.0039). Large-dose cyclic TPD is a useful prescription of PD for increasing Kt/V and fluid removal.
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http://dx.doi.org/10.1111/1744-9987.12765DOI Listing
April 2019

Biomarker discovery by integrated joint non-negative matrix factorization and pathway signature analyses.

Sci Rep 2018 06 27;8(1):9743. Epub 2018 Jun 27.

Human Genome Center, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan.

Predictive biomarkers are important for selecting appropriate patients for particular treatments. Comprehensive genomic, transcriptomic, and pharmacological data provide clues for understanding relationships between biomarkers and drugs. However, it is still difficult to mine biologically meaningful biomarkers from multi-omics data. Here, we developed an approach for mining multi-omics cell line data by integrating joint non-negative matrix factorization (JNMF) and pathway signature analyses to identify candidate biomarkers. The JNMF detected known associations between biomarkers and drugs such as BRAF mutation with PLX4720 and HER2 amplification with lapatinib. Furthermore, we observed that tumours with both BRAF mutation and MITF activation were more sensitive to BRAF inhibitors compared to tumours with BRAF mutation without MITF activation. Therefore, activation of the BRAF/MITF axis seems to be a more appropriate biomarker for predicting the efficacy of a BRAF inhibitor than the conventional biomarker of BRAF mutation alone. Our biomarker discovery scheme represents an integration of JNMF multi-omics clustering and multi-layer interpretation based on pathway gene signature analyses. This approach is also expected to be useful for establishing drug development strategies, identifying pharmacodynamic biomarkers, in mode of action analysis, as well as for mining drug response data in a clinical setting.
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http://dx.doi.org/10.1038/s41598-018-28066-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021419PMC
June 2018

Congenital chloride diarrhea needs to be distinguished from Bartter and Gitelman syndrome.

J Hum Genet 2018 Jul 30;63(8):887-892. Epub 2018 May 30.

Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.

Pseudo-Bartter/Gitelman syndrome (p-BS/GS) encompasses a clinically heterogeneous group of inherited or acquired disorders similar to Bartter syndrome (BS) or Gitelman syndrome (GS), both renal salt-losing tubulopathies. Phenotypic overlap frequently occurs between p-BS/GS and BS/GS, which are difficult to diagnose based on their clinical presentation and require genetic tests for accurate diagnosis. In addition, p-BS/GS can occur as a result of other inherited diseases such as cystic fibrosis, autosomal dominant hypocalcemia, Dent disease, or congenital chloride diarrhea (CCD). However, the detection of the variants in genes other than known BS/GS-causing genes by conventional Sanger sequencing requires substantial time and resources. We studied 27 cases clinically diagnosed with BS/GS, but with negative genetic tests for known BS/GS genes. We conducted targeted sequencing for 22 genes including genes responsible for tubulopathies and other inherited diseases manifesting with p-BS/GS symptoms. We detected the SLC26A3 gene variants responsible for CCD in two patients. In Patient 1, we found the SLC26A3 compound heterozygous variants: c.354delC and c.1008insT. In Patient 2, we identified the compound heterozygous variants: c.877G > A, p.(Glu293Lys), and c.1008insT. Our results suggest that a comprehensive genetic screening system using targeted sequencing is useful for the diagnosis of patients with p-BS/GS with alternative genetic origins.
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http://dx.doi.org/10.1038/s10038-018-0470-7DOI Listing
July 2018

3D culture system containing gellan gum restores oncogene dependence in ROS1 rearrangements non-small cell lung cancer.

Biochem Biophys Res Commun 2018 06 10;501(2):527-533. Epub 2018 May 10.

Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31, Ariake, Koto-ku, Tokyo, 135-8550, Japan. Electronic address:

The ROS1 fusion gene has been identified in approximately 1% of non-small cell lung cancer (NSCLC) cases. Several clinical studies have highlighted ROS1 as a promising therapeutic target because crizotinib, a multi-targeted drug against ROS1, ALK, and the MET proto-oncogene, has elicited remarkable responses in ROS1-rearrangements NSCLC. However, acquired resistance mediated by ROS1 kinase domain mutations has been identified and a system to assess ROS1 inhibitors for these resistant mutations is necessary for the promotion of drug development. Publicly available NSCLC cell lines harboring the ROS1 fusion gene are limited to only HCC78 cells carrying SLC34A2-ROS1. This cell line exhibits resistance to ROS1 inhibitors through activation of the EGFR pathway, although the cells were established from ROS1-TKI naïve pleural effusion. Here, we demonstrate that 3D culture with gellan gum can restore the ROS1 oncogene dependence of HCC78 cells by upregulating the expression of the ROS1 fusion gene and reducing the activity of the EGFR pathway. Moreover, we established the HCC78xe3 cell line, a subclone of the HCC78 cell line, by repeated in vitro cultures from tumor xenografts and created xenograft tumors three times using in vitro cultured cells. This eventually enabled us to engraft and stably grow the cells in vivo, and subsequently evaluate various ROS1 inhibitors against HCC78xe3 cells by overexpressing crizotinib-resistant mutations in the ROS1 kinase domain including G2032R and D2033 N. We newly found that lorlatinib, a next generation ROS1/ALK inhibitor, remain the activity against D2033 N mutation. Furthermore, we demonstrated that HCC78xe3 cells expressing SLC34A2-ROS1 G2032R, and D2033 N, but not wild type (WT) cells, were resistant to crizotinib in vivo. Taken together, our data suggested that 3D cultures of HCC78 might reflect the features in patients and this new system will be a useful tool for evaluating ROS1 inhibitors.
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http://dx.doi.org/10.1016/j.bbrc.2018.05.031DOI Listing
June 2018

Identification of Mutation Accumulation as Resistance Mechanism Emerging in First-Line Osimertinib Treatment.

J Thorac Oncol 2018 07 24;13(7):915-925. Epub 2018 Apr 24.

Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan. Electronic address:

Introduction: The survival of patients with EGFR mutation-positive lung cancer has dramatically improved since the introduction of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Recently, osimertinib showed significantly prolonged progression-free survival than first-generation EGFR-TKI in first-line treatment, suggesting that a paradigm change that would move osimetinib to first-line treatment is indicated. We performed N-ethyl-N-nitrosourea (ENU) mutagenesis screening to uncover the resistant mechanism in first- and second-line osimertinib treatment.

Methods: Ba/F3 cells harboring EGFR activating-mutation with or without secondary resistant mutation were exposed to ENU for 24 hours to introduce random mutations and selected with gefitinib, afatinib, or osimertinib. Mutations of emerging resistant cells were assessed.

Results: The resistance of T790M and C797S to gefitinib and osimertinib, respectively, was prevalent in the mutagenesis screening with the Ba/F3 cells harboring activating-mutation alone. From C797S/activating-mutation expressing Ba/F3, the additional T790M was a major resistant mechanism in gefitinib and afatinib selection and the additional T854A and L792H were minor resistance mechanisms only in afatinib selection. However, the additional T854A or L792H mediated resistance to all classes of EGFR-TKI. Surprisingly, no resistant clone due to secondary mutation emerged from activating-mutation alone in the gefitinib + osimertinib selection.

Conclusions: We showed the resistance mechanism to EGFR-TKI focusing on first- and second-line osimertinib using ENU mutagenesis screening. Additional T854A and L792H on C797S/activating-mutation were found as afatinib resistance and not as gefitinib resistance. Thus, compared to afatinib, the first-generation EGFR-TKI might be preferable as second-line treatment to C797S/activating-mutation emerging after first-line osimertinib treatment.
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http://dx.doi.org/10.1016/j.jtho.2018.04.005DOI Listing
July 2018