ALBERT

Description: To elucidate the molecular pathogenesis of pediatric germ cell tumors (GCTs), we performed DNA methylation array analysis, whole transcriptome sequencing, targeted capture sequencing, and single-nucleotide polymorphism array analysis using 51 GCT samples (25 female, 26 male), including 6 germinomas, 2 embryonal carcinomas, 4 immature teratomas, 3 mature teratomas, 30 yolk sac tumors, and 6 mixed germ cell tumors. Among the 51 samples, 11 were from infants, 23 were from young children, and 17 were from those aged ≥10 years. Sixteen of the 51 samples developed in the extragonadal regions. Germinomas showed upregulation of pluripotent genes and global hypomethylation. Pluripotent genes were also highly expressed in embryonal carcinomas. These genes may play essential roles in embryonal carcinomas given that their binding sites are hypomethylated. Yolk sac tumors exhibited overexpression of endodermal genes, such as GATA6 and FOXA2, the binding sites of which were hypomethylated. Interestingly, infant yolk sac tumors had different DNA methylation patterns from those observed in older children. Teratomas had higher expression of ectodermal genes, suggesting a tridermal nature. Based on our results, we suggest that KIT, TNFRSF8, and ERBB4 may be suitable targets for the treatment of germinoma, embryonal carcinomas, and yolk sac tumors, respectively.

Description: Introduction TP53 mutations in relapsed cases with pediatric acute lymphoblastic leukemia have been implicated in poor clinical outcomes. However, the prevalence and clinical significance of TP53 mutations at diagnosis have not been fully investigated. Such knowledge is essential for the care of patients, because treatment intensity is tailored to predictive prognosis, where increased attention has been directed toward de-escalation of treatment for the problem of long term effects and second malignancies in childhood cancer survivors. Methods Mutation status of TP53 was detected by targeted-capture sequencing of TP53 coding regions in 1,003 children with B-precursor ALL who had been treated in either of the two prospective clinical trials, JACLS (Japan Association of Childhood Leukemia Study) ALL-02 and TCCSG (Tokyo Children's Cancer Study Group) L04-16. Detection of common fusion genes, including BCR-ABL, ETV6-RUNX1, MLL-AF4, MLL-ENL, MLL-AF9, and TCF3-PBX1, were performed using qPCR assays. We designed SNP baits to analyze copy number status of chromosome 17, and also captured 662 probes tiling the entire IgH enhancer locus to identify IGH-DUX4 rearrangement. Result In total, 36 different non-silent coding TP53 mutations were identified in 30 (3%) patients, including 22 missense, 7 frameshift indel, 5 in-frame indel, and 2 nonsense mutations. All missense mutations were found in the core DNA-binding domain (n=21), except for one mutation, which affected the tetramerization motif. Variant allele frequencies (VAF) of TP53 mutations varied from 3% to 97% with 14 mutations showing 〈 10% VAFs. Showing a significant correlation with mutated TP53 (Odds ratio 20: 95%CI 6.4-61, P

Description: Introduction NOTCH1 and FBXW7 alterations leading to aberrant activation of NOTCH1 signaling, classified into two patterns; ligand-independent activation (LIA) and impaired degradation (ID) of NOTCH1. In general, activation of NOTCH1 axis is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), though comprehensive studies regarding subclonal mutations inducing NOTCH1 activation are still elusive. In the present study, we explored the clinicopathological relevance of NOTCH1/FBXW7 aberrations considering subclonal alterations. Methods A total of 176 cases with pediatric T-ALL were enrolled in this study. We reanalyzed our previous data of targeted-capture sequencing (n=176) for 158 ALL-related genes/regions and combined with previous expression profiling data based on whole transcriptome sequencing (WTS; n=121). We defined as a subclonal mutation when variant allele frequency was below 0.15 and/or multiple alterations were found within the same pattern of NOTCH1 activation (LIA or ID). All patients were received Berlin-Frankfurt-Münster based chemotherapies with non-minimal residual disease (MRD) based risk stratification, which were mainly offered from the Tokyo Children's Cancer Study Group (TCCSG) and the Japan Association of Childhood Leukemia Study (JACLS). Results In total, we detected aberrations activating NOTCH1 signaling in 81.3% (143/176) of cases including subclonal mutations. Subclonal alterations were observed in 26.7% (n=47). Single nucleotide variations in the heterodimerization domain (HD-SNV) were the most frequent (43.2%; n=76), followed by PEST domain mutations (33.0%; n=58), FBXW7 mutations (26.1%; n=46), non-frameshift indels of NOTCH1 (19.9%; n=35), and in-frame internal duplication known as juxta-membrane expansion (6.3%; n=11). Amplification of NOTCH1 region and 5' NOTCH1 deletion were not detected in our cohort. Both LIA and ID patterns were detected in 43.2% (n=76). Most mutations were mutually exclusive within each LIA and ID pattern. Intriguingly, we detected four (2.3%) internal deletion of NOTCH1 gene (DEL; missing exon 3-27 (DEL3) or 21-27 (DEL21)), three cases (1.7%) of SNV at 3' untranslated region, and two (1.1%) SEC16A-NOTCH1 fusions. These alterations were previously reported to activate NOTCH1 signaling in breast cancer or chronic lymphoblastic leukemia, except for DEL21. We confirmed that DEL21 strongly activates NOTCH1 signaling by luciferase reporter assay (over 100 times compared to wild type NOTCH1). As previously reported in DEL3 and CUTLL cell line, transcripts might initiate at methionine 1737 located within the NOTCH1 transmembrane domain and seem to be sensitive to γ-secretase inhibitors. Analysis of frequency of detected NOTCH1 activating alterations in each previously reported WTS-based cluster (ETP, SPI1, TLX, TAL1-RA, and TAL1-RB) revealed that alterations were frequently detected in TLX (100%; 24/24) and TAL1-RB (95.1%; 39/41), whereas less frequent in TAL1-RA (61.1%; 11/18). In TAL1-RA, all SEC16A-NOTCH1 fusions were observed despite significantly low rate of HD-SNV (11.1%; 2/18). In SPI1 cluster, PEST domain alterations were frequently detected (71.4%; 5/7). Importantly, cases harboring subclonal NOTCH1/FBXW7 alterations showed significantly worse outcome (log-rank P = 0.01), although there was no prognostic difference between cases with and without NOTCH1/FBXW7 mutations. Conclusions We observed NOTCH1 activating alterations in 81.3% of pediatric T-ALL cases and detected rare internal deletion of NOTCH1 gene and NOTCH1 fusions recurrently in T-ALL. Furthermore, the presence of subclonal NOTCH1/FBXW7 mutations might be relevant to unfavorable outcome. Despite several limitations such as non-MRD based treatment, our results might be useful for developing a new anti-NOTCH1 therapeutic strategy for pediatric T-ALL patients. Disclosures No relevant conflicts of interest to declare.

Description: Background Acute lymphoblastic leukemia (ALL) in Down syndrome (DS) have uncommon genetic alterations such as mutations of JAK2, RAS, and overexpressions of CRLF2. These findings suggest DS-ALL may have unique biological features compared with non-DS-ALL. While recent studies implicated HMGN1 or DYRK1A in chromosome 21 were associated with molecular pathogenesis of DS-ALL, it remains to be elucidated what predispose DS children to develop ALL. Materials and Methods We performed whole transcriptome sequencing, targeted deep sequencing, and SNP array analysis in 25 DS-ALL samples, which included four ETV6-RUNX1 fusions and one high hyperdiploid. To compare with DS-ALL, we also performed whole transcriptome sequencing and whole exome sequencing to 118 non-DS-ALL samples, which included several subtypes such as ETV6-RUNX1 or BCR-ABL1. To cluster gene expression profiling, we applied the hierarchical clustering method. The detection of Ph-like signatures was performed by the hierarchical clustering by the gene set reported by Harvey. Results In expression analysis, we identified 19 fusions in 25 DS-ALL samples. These fusions included 15 recurrent fusions in pediatric BCP-ALL and 4 novel fusions, which including SSBP3-DHCR24, PDGFA-TTYH3, and NIN-NDUFA6. In novel fusions, PDGFA-TTYH3 fusions were detected in two DS-ALL samples. The hierarchical clustering analysis (Figure 1) combining 25 DS-ALL with 123 non-DS ALL samples. In our cohort, we defined samples with PAX5 alteration only such as a mutation or fusion as PAX5-altered. This clustering revealed ALL samples were divided into six clusters (cluster E1 to E6). Among six clusters, DS-ALL samples were divided into four clusters. In these four clusters, chi-square test revealed the significant enrichment of DS-ALL in E6 cluster. Importantly, our expression analysis revealed DS-ALL samples were highly heterogeneous and had the same expression pattern corresponding to each subtype same as non-DS-ALL. Cluster E3 included most samples with PAX5 fusions. All samples with ETV6-RUNX1 fusions were classified into cluster E4. Most samples of high hyperdiploid were classified into cluster E5. Cluster E6 was characterized by BCR-ABL1 fusions and Ph-like signatures. We detected 21 samples had Ph-like signatures, which included seven DS-ALL samples and 14 non-DS-ALL samples. Though we also analyzed differentially expressed genes between DS-ALL and non-DS-ALL, no genes on chromosome 21 such as HNGN1 or DYRK1A was significantly expressed. To investigate a relation between expression and genomic status, we further searched mutational analysis and copy number analysis (Figure 2). In 25 DS-ALL samples, six samples revealed JAK2 mutations and CRLF2 fusions. Interestingly, all of these six samples had Ph-like signatures. In cluster E5, one non-DS-ALL sample revealed JAK2 mutation and CRLF2 fusion and this particular sample was expected to have the Ph-like signature. To detect other Ph-like samples, we performed hierarchical clustering of 143 ALL samples based on the genes with a significantly (adjusted P value

Description: T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL). Recent genome-wide approach revealed frequent NOTCH1 and FBXW7 oncogenic mutations in T-ALL. In addition, previous whole-exome sequencing disclosed novel CNOT3 mutations in approximately 10% of adult T-ALL cases, and thus, CNOT3 is thought to be one of the novel tumor suppressor gene for adult T-ALL. However, somatic mutations in these genes have been found in a fraction of childhood T-ALL, suggesting that the existence of other genetic pathogenesis. Although chromosomal translocations are the most frequent genetic abnormalities detected in other types of leukemia, recurrent translocations except for SIL-TAL1 rearrangement have been poorly defined in T-ALL. To discover driver mutations or fusion genes which involved in the pathogenesis of pediatric T-ALL and to identify novel prognostic markers of childhood T-ALL, we performed whole-exome sequencing (WES) and transcriptome sequencing (WTS) in 25 cases with T-ALL. Diagnostic total DNA from 25 cases and RNA from 15 cases were analyzed for both WES and WTS, and 8 relapsed samples were also analyzed for WES. Median age at diagnosis was 9 years old (1–15), and male to female ratio was 20 to 5. Libraries for WES and WTS were generated using the SureSelect (Agilent) or TruSeq RNA Sample Preparation kit (Illumina), respectively. High throughput sequencing was performed using the Illumina HiSeq 2000 platform. To detect somatic mutations or fusion transcripts, we used our pipeline “Genomon-exome” and “Genomon-fusion” algorithm. Subsequently, somatic mutations were validated using deep amplicon sequencing. Candidate fusion transcripts were validated by reverse - transcription polymerase-chain-reaction (RT-PCR) and Sanger sequencing. Most frequent mutation was NOTCH1, which was detected in 52% (13/25) by WES. FBXW7 mutations were also frequently found in 28% (7/25), and 43 % (3/7) were compound heterozygous mutations. In those 6 cases, only one case with FBWX7 mutation had a NOTCH1 mutation. CNOT3 mutations were reported to be frequent in adult T-ALL; however we found only 2 cases with CNOT3 mutations (8.0%). In addition, PHF6 mutation, which is known as X-linked tumor suppressor gene in T-ALL, was recurrently detected in 4 cases (16%). Other recurrent mutations were shared between 2 cases, respectively. We identified previously known fusion genes, such as MLL-ENL and FGFROP1-FGFR1 in 2 cases. MLL-ENL is one of the frequent translocation for infant multilineage leukemia (MLL), but also reported in non-infant B cell precursor ALL or T-ALL. FGFR1OP is ubiquitously expressed, and the predicted chimeric FGFR1OP-FGFR1 protein contains the catalytic domain of FGFR1. It is thought to be promote hematopoietic stem cell proliferation and leukemogenesis through a constitutive phosphorylation and activation of the downstream pathway of FGFR1. In conclusion, although NOTCH1 and FBXW7 mutations were relatively frequently detected in our series, we could not detect frequent additional mutations in this study. Consistent with other reports, frequent translocations were not observed in T-ALL, suggesting the genetic differences between T-ALL and other hematological malignancies. Further studies will be necessary to unravel oncogenic mechanisms that implicated in new therapeutic strategy for pediatric T-ALL. Disclosures No relevant conflicts of interest to declare.

Description: Background Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer accounting for 10 to 15% of newly diagnosed pediatric ALL cases, and is associated with a poor treatment outcome compared with B-cell ALL especially in relapsed cases despite recent improvement with an intensive treatment. TAL1 deletion on chromosome 1p33 which is also known as STIL-TAL1, is found in 20-25% of cases with T-ALL, which results in overexpression of TAL1 under the control of the promoter of STIL located next to TAL1. Recently, TAL1 super enhancer aberration (TAL1-SE) was reported, which introduce binding motifs for the MYB transcription factor in a noncoding region. This somatic mutation results in aberrant expression of TAL1 without STIL-TAL1. Clinical features of TAL1-SE and its difference with STIL-TAL1 cases have been poorly studied, and this could be a new instrument of treatment stratification in T-ALL. Methods Targeted capture sequencing for coding regions of 151 genes using a custom-made bait including the TAL1-SE region was performed in 132 Japanese T-ALL cases under 15 years old to detect somatic mutations. For the further analysis, whole transcriptome sequencing (WTS) was performed in 88 cases. Screening for STIL-TAL1 fusion by reverse transcription PCR was also performed in additional 44 cases. All cases of TAL1-SE and STIL-TAL1 were validated by Sanger sequencing. Our pipelines,"Genomon 2" algorithm, were used for detection of somatic mutations and fusions. Most samples of DNA and RNA were offered from Tokyo Children's Cancer Study Group (TCCSG), and available clinical data were mainly based on the TCCSG clinical study treated with BFM-based ALL treatment protocol. Results TAL1-SE and STIL-TAL1 were found mutually exclusive in 6 (4.5%) and 24 cases (18.1%), respectively. All patients with TAL1-SE were under 10 years old. TAL1-SE cases showed a favorable 3-year overall survival (3y-OS) compared with STIL-TAL1 cases which were reported to show a good prognosis (83.3% vs 85.9% respectively, p = 0.71). However, 3-year disease free survival (3y-DFS) was 22.2% and 72.3% respectively (p = 0.04), indicating higher relapse rate in TAL1-SE cases, and they were probably rescued by second line treatment. Patients with STIL-TAL1 showed significantly poorer prednisolone response (over 1,000 /µl of blast at day 8, p 〈 0.05), though they showed favorable 3y-OS, which might be resulted from frequent high WBC counts at diagnosis (〉 100×106/l, 59%). Comparison of TAL1 expression using read counts of WTS revealed significantly higher TAL1 expression in TAL1-SE cases than STIL-TAL1 cases (p 〈 0.05). To reveal the difference of gene expression profile between STIL-TAL1 and TAL1-SE, unsupervised consensus clustering of gene expression data in 24 cases performed WTS (6 cases of TAL1-SE and 18 cases of STIL-TAL1) divided into 2 clusters. All cases with TAL1-SE were grouped into the same cluster with 10 STIL-TAL1 cases (Cluster 1). The other 8 STIL-TAL1 cases were classified as Cluster 2. A genetic landscape of these 24 cases showed more frequent NOTCH1 and/or FBXW7 mutations in Cluster 1 (87.5% vs 37.5%, p = 0.02), whereas mutations in genes encoding transcription factor such as RUNX1 and BCL11B were found only in Cluster 1 (n = 1, n = 3, respectively). There was a tendency of better 3y-OS and 3y-DFS in Cluster 2 (73.9% vs 100%, p = 0.14, 55.6% vs 85.7%, p = 0.15, respectively). Conclusion TAL1-SE and STIL-TAL1 showed a relatively good prognosis, though high relapse rate in TAL1-SE and a poor prednisolone response in STIL-TAL1 were observed. STIL-TAL1 cases were biologically divided into 2 groups based on the expression profile (with TAL1-SE and STIL-TAL1 only), and these 2 groups showed different clinical and genetic features such as 3y-OS or frequency of NOTCH1/FBXW7 mutations. Our findings illustrate the clinicopathological differences between STIL-TAL1 and TAL1-SE cases, and thus, it might be helpful for development a new therapeutic strategy for these patients with TAL1 overexpression T-ALL. Disclosures Kataoka: Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Description: T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL). Although gene fusions generated through chromosomal translocations, deletions, and inversions are the most frequent genetic abnormalities detected in other types of leukemia, recurrent gene fusions except for SIL-TAL1 have been poorly defined in T-ALL. To discover driver mutations or fusion genes, which involved in the pathogenesis of childhood T-ALL and to identify novel prognostic markers of childhood T-ALL, we performed whole-exome sequencing (WES) and transcriptome sequencing (WTS) in 31 and 46 cases, respectively. We also performed SNP array karyotyping in 46 cases. To detect somatic mutations or fusion transcripts, we used our pipeline "Genomon-exome" and "Genomon-fusion" algorithm. Subsequently, somatic mutations were validated using deep amplicon sequencing. Candidate fusion transcripts were validated by reverse - transcription polymerase-chain-reaction and Sanger sequencing.Recurrent mutations observed in more than 3 cases were detected in NOTCH1 (n = 18, 58%), FBXW7 (n = 7, 23%), PHF6 (n = 5, 16%), GATA3 (n = 4, 13%), MYB (n = 3, 10%), and NRAS (n = 3, 10%), respectively. We identified previously known fusion genes, such as MLL-ENL (n = 2), CALM-AF10 (n = 2), NUP214-ABL1 (n = 1) and FGFROP1-FGFR1 (n = 1). MLL-ENL is one of the frequent translocation in infant multilineage leukemia, but also reported in non-infant B cell precursor ALL and T-ALL. FGFR1OP is ubiquitously expressed, and the predicted chimeric FGFR1OP-FGFR1 protein contains the catalytic domain of FGFR1. It is thought to promote hematopoietic stem cell proliferation and leukemogenesis through a constitutive phosphorylation and activation of the downstream pathway of FGFR1. CALM-AF10 leukemia is reported to increase HOXA cluster gene transcription, we could also confirm elevated HOXA genes expression by FPKM value. Four SIL-TAL1 fusions were detected in our cohort. Recently, a novel mutation in TAL1 enhancer region which introduce de novo MYB biding site has been reported. Since this abnormality lead high expression of TAL1, we also analyzed expression data obtained from WTS. Among 46 specimens, 19 samples showed high expression of TAL1 (FPKM value ≥5). In those cases, 4 cases had SIL-TAL1 fusions (8%), and 3 cases (6%) had insertions in enhancer region of TAL1. Subsequent analysis using Gene Set Enrich Analysis (GSEA) between TAL1 high and low expression samples revealed that "LEE_EARLY_T_LYMPHOCYTE_UP" was enriched in TAL1 high expression samples (Enrichment score = 0.73, FDR = 0.073). This gene set includes genes up-regulated at early stages of progenitor T lymphocyte maturation compared to the late stages, and MYB was included in this gene set. Intriguingly, MYB mutation samples were not represented TAL1 high expression. TAL1 related rearrangement or enhancer insertion was not detected in the rest of 12 cases with TAL1 high expression, suggesting that other mechanisms of TAL1 high expression might be exist. In conclusion, although NOTCH1 and FBXW7 mutations were relatively frequently detected in our series, we also found recurrent MYB mutations. SIL-TAL1 was known as most frequent rearrangement, TAL1 enhancer insertions were also frequent in TAL1 overexpressed samples. TAL1 enhancer insertion and MYB mutation was exclusive, suggesting that TAL1 and MYB have a key role in childhood T-ALL. Consistent with other reports, frequent translocations were not observed in T-ALL, suggesting the genetic differences between T-ALL and other hematological malignancies. Further studies will be necessary to unravel oncogenic mechanisms that implicated in new therapeutic strategy for childhood T-ALL. Disclosures No relevant conflicts of interest to declare.

Description: T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL), arising from the malignant transformation of hematopoietic progenitors primed toward T cell development, as result of a multistep oncogenic process. However, since the prognostic significance of these genetic alterations in pediatric T-ALL is not clear, genetic basis which contributes aggressive phenotype or progression of pediatric T-ALL is still to be elucidated. Therefore, to discover driver genetic events, which involved in the aggressive phenotype of pediatric T-ALL and to identify it's novel prognostic markers, we performed integrated genetic analysis in a large cohort of T-ALL case. Our cohorts included samples from Tokyo Children's Cancer Study Group (TCCSG) and Japan Association of Childhood Leukemia Study (JACLS). Whole transcriptome sequencing (WTS) was performed in 123 cases. Representative recurrent fusion genes were as follows, SIL-TAL1 (n=25), MLL-ENL (n=5), PICALM-MLLT10 (n=5), and NUP214-ABL1 (n=2). Intriguingly, novel recurrent in-frame SPI1 fusions (STMN1-SPI1 n=2; TCF7-SPI1 n=5) were detected, and RT-PCR analysis in additional 60 cases revealed other 2 TCF7-SPI1 fusions. Thus, SPI1 fusions accounted for 4% of pediatric T-ALL/LBL. Expression data of WTS revealed cases with SPI1 fusion showed significantly higher expression of SPI1 compared to cases without SPI1 fusion, implicating that aberrant high expression of SPI1 involved in leukemogenesis. To address the functional activities of SPI1 fusions, we performed luciferase assay using the reporter vector contained the CSF1 promoter region with SPI1 binding site. Transient transfection of Hela cells with the SPI1 fusions expression vectors as well as the wild type SPI1 expression vector showed strikingly high levels of transcription of the reporter genes, as compared to transfection with the empty expression vector, indicating that both SPI1 fusions have transcriptional activities. Next, to analyze the leukemogenic potential of SPI1 fusions in vitro, we transduced fusions cDNA into mouse double negative T-cells. Since p16(CDKN2A) is frequently silenced in T-ALL, we also used p16 null double negative T-cells. Both wild-type and p16 null double negative T-cells expressing SPI1 fusions showed increased cell proliferation compared to the MOCK cells. We further evaluated the impact of SPI1 fusions on T cells differentiation. TCF7-SPI1 or MOCK vector was transduced mononuclear cells isolated from mouse bone marrow. These cells were cultured under stimulating factors, such as IL6 and TPO for 3 days, and then transplanted into the irradiated mouse. Subsequently, 6 week after transplantation, FACS analysis was performed. Of interest, significantly high population of cells expressing TCF7-SPI1 was observed in the immature single positive stage, implicating that their differentiation was impaired at the pre-T cell stage. These results indicate that novel SPI1 fusions have a potential leukemogenic effect in pediatric T-ALL. We defined SPI1 overexpression cases as outliers of SPI1 expression, resulting in extremely poor prognosis (log-rank p = 1.9 ×10-6). Of note, significant poor outcome was confirmed by univariate and multivariate analysis in cases with SPI1 overexpression cases (log-rank p = 9.3 ×10-6, and p = 2.0 ×10-6, respectively). In conclusion, SPI1 fusions expressing cells expanded and they remained at an immature stage, implicating a potential leukemogenic activity of these fusions. Not only the cases with SPI1 fusions, but also the cases with high SPI1 expression without fusions showed extremely poor prognosis, suggesting the prognostic value of aberrant SPI1 expression in pediatric T-ALL. Although it remains unclear, why cases with SPI1 fusions/high SPI1 expression have a poor prognosis, our results indicate that these cases are genetically distinct subgroup from other pediatric T-ALL. Disclosures Kataoka: Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.