Daeyoon Kim

Epidermal regeneration by ent-16α, 17-dihydroxy-kauran-19-oic acid isolated from Siegesbeckia pubescens.

Objectives:  Keratinocyte stem/progenitor cells (KSCs) are known to regenerate epidermal tissue which they perform through to their great regenerative capacity.

Revealing Genomic Profile That Underlies Tropism of Myeloma Cells Using Whole Exome Sequencing.

Background. Previously we established two cell lines (SNU_MM1393_BM and SNU_MM1393_SC) from different tissues (bone marrow and subcutis) of mice which were injected with single patients myeloma sample. We tried to define genetic changes specific for each cell line using whole exome sequencing (WES). Materials and Methods. We extracted DNA from SNU_MM1393_BM and SNU_MM1393_SC and performed WES. For single nucleotide variants (SNV) calling, we used Varscan2. Annotation of mutation was performed using ANNOVAR. Results. When calling of somatic mutations was performed, 68 genes were nonsynonymously mutated only in SNU_MM1393_SC, while 136 genes were nonsynonymously mutated only in SNU_MM1393_BM. KIAA1199, FRY, AP3B2, and OPTC were representative genes specifically mutated in SNU_MM1393_SC. When comparison analysis was performed using TCGA data, mutational pattern of SNU_MM1393_SC resembled that of melanoma mostly. Pathway analysis using KEGG database showed that mutated genes specific of SNU_MM1393_BM were related to differentiation, while those of SNU_MM1393_SC were related to tumorigenesis. Conclusion. We found out genetic changes that underlie tropism of myeloma cells using WES. Genetic signature of cutaneous plasmacytoma shares that of melanoma implying common mechanism for skin tropism. KIAA1199, FRY, AP3B2, and OPTC are candidate genes for skin tropism of cancers.

Oncogenic effects of germline mutations in lysosomal storage disease genes

Introduction Lysosomal storage diseases (LSDs) comprise inborn metabolic disorders caused by mutations in genes related to lysosomal function. As already observed in certain LSDs, the accumulation of macromolecules caused by lysosomal dysfunction may facilitate carcinogenesis. Methods Using whole genome sequencing data from the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Network (Pan-Cancer cohort) and the 1000 Genomes project (1000 Genomes cohort), we analyzed the association between potentially pathogenic variants (PPVs) in 42 LSD genes and cancer. We evaluated age at diagnosis of cancer and patterns of somatic mutation and gene expression according to the PPV carrier status. Results PPV prevalence in the Pan-Cancer cohort was significantly higher than that of the 1000 Genomes cohort (20.7% versus 13.5%, P=8.7×10−12). Cancer risk was increased in individuals with a greater number of PPVs. Population structure-adjusted SKAT-O analysis revealed 37 significantly associated cancer type-LSD gene pairs. These results were validated using the ExAC cohort as an independent control. Cancer developed earlier in carriers of PPVs in pancreatic adenocarcinoma (MAN2B1, GALNS, and GUSB), skin cancer (NPC2), and chronic myeloid disorder (SGSH) as well as in the Pan-Cancer cohort. Analysis of RNA-Seq data from the pancreatic cancer cohort revealed 508 genes differentially expressed according to the PPV carrier status, which were highly enriched in the known core signaling pathways of pancreatic cancer. Conclusion Carriers of germline PPVs in LSD genes have an increased incidence of cancer. The available therapeutic options to restore lysosomal function suggest the potential of personalized cancer prevention for these patients.

Abstract 2446: Integrated analysis of somatic mutations in subcutaneous panniculitis-like T-cell lymphoma by whole-exome and -transcriptome sequencing

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rarely differentiated form of non-Hodgkin lymphoma. This uncommon disease is triggered by preferential lymphoma infiltration into subcutaneous adipose tissue, and nodule formation in the fatty tissue. Consequently, the early stage symptoms of SPTCL is found as multiple subcutaneous nodules beneath the skin. Due to the low incidence rate and indolent progression of the disease, the genetic alterations associated with SPTCL still remains poorly characterized. Genetic profiling of SPTCL by integrated whole-exome and -transcriptome sequencing will augment the comprehensive characterization of somatic mutations in SPTCL. To investigate the genetic alterations associated with the incidence of SPTCL, we performed whole-exome sequencing of both biopsy sample collected from the patient’s left shoulder lesion, and matched saliva sample to 200X and 100X read depth of coverage, respectively. Whole-transcriptome sequencing of the tumor sample was also performed for expression level analysis; gene fusions are also explored by TopHat-fusion-post. Somatic mutations were precisely detected by our internally developed somatic variant caller, which is an adjusted version of VarScan2 with additional filters based on thresholds of p-value and odds ratio. Overall, we discovered a total of 158 somatic variants: 139 SNVs and 19 indels, by using somatic variant detection algorithm of our own. By applying the additional filters to these variants, two genes were remarkably identified: SQSTM1 and BAGE3. SQSTM1 (sequestosome 1), also known as ubiquitin-binding protein p62, encodes proteins that regulate ubiquitination, autophagy, and activation of NFkB1. Through cBioPortal database search, SQSTM1 found to be highly amplified across the various cancer types, such as neuroendocrine prostate cancer (NEPC), kidney renal clear cell carcinoma (RCC), and pancreatic cancer. On the other hand, BAGE3, B melanoma antigen 3, was reported as a candidate gene encoding tumor antigens. Gene fusions were identified by comparing the number of spanning reads and mate pairs, and fusion of NOL7 and RANBP9 on chromosome 6 showed the highest fusion score of 604.45. Comprehensive characterization of the initiation, progression, and relapse of subcutaneous panniculitis-like T-cell lymphoma is yet thoroughly understood. Hence, establishing the genetic basis and profiling the genomic landscape of SPTCL will broaden our understanding of SPTCL, and enhance the therapeutic effectiveness in the pre-diagnosis and treatment of the disease, as well as targeted gene therapies. Furthermore, to strengthen the statistical power of the somatic mutation analysis, we are planning to expand the cohort and conduct functional validation study of selected genes by using secured paraffin blocks of SPTCL patients. Citation Format: Hyojin Song, Youngil Koh, Daeyoon Kim, Hongseok Yun, Choong-Hyun Sun, Hogune Im, Dong-Yeop Shin, Sung-Soo Yoon. Integrated analysis of somatic mutations in subcutaneous panniculitis-like T-cell lymphoma by whole-exome and -transcriptome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2446. doi:10.1158/1538-7445.AM2017-2446

Abstract 2582: KIRnome: KIR genotyping for whole genome/exome sequencing data

In this study, we tried to develop valid KIR calling algorithm from WES/WGS data. As a consequence, here we suggest KIRnome (KIR typing for whole genome and exome sequencing) which is a KIR typing method for applying sequencing data such as WES/WGS. A total of 71 sequencing data (18 WES and 53 WGS data, respectively) and matched experimentally validated KIR genotype data are used to train and validate. Before developing this method, two types of allele references (genomic and coding, here after called gSeq and cSeq) are constructed by allele sequence of IPD/KIR. KIRnome consists of two part, first part is calculating normalized depth (here after called depth) of 16 KIR genes and considering allele reference length and second is estimating KIR type based on depth. In general, we get higher normalized depth when using genomic references for WGS data and using coding references for WES data. This coincides with scheme KIRnome and the characteristics of WGS and WES data. Given 71 samples of 3 data sets, we evaluated KIR genotyping performance of KIRnome per each KIR gene. For all 16 KIR genes, KIRnome shows >=98.5% accuracy. When KIRnome performance was tested only in 13 WES samples, the accuracy slightly drops to 94%. We assume this is attributable to relatively small number of the samples. In conclusion, we developed a novel and unique method named KIRnome for KIR typing from NGS data. KIRnome could determine KIR genotype accurately from WES and WGS data respectively. We expect KIRnome would facilitate revealation of immunogenetic facts in various disease. Moreover, future generation of KIR specific NGS data and improved reference sequence information of KIR would enable KIRnome to type KIR at allele level in a near future. Citation Format: Daeyoon Kim, Sung-Soo Yoon, Youngil Koh, Su Yeon Lee, Hongseok Yun, Sunghoon Cho, Hyung-Lae Kim. KIRnome: KIR genotyping for whole genome/exome sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2582. doi:10.1158/1538-7445.AM2017-2582

Abstract 384: The implications of splicing variant of AIMP2 lacking exon 2 among various cancer types: An analysis of the ICGC/TCGA database and clinical validation

Aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMP) is the multiple tRNA synthetase complex protein called the multi-tRNA complex (MRC). In cancer, the splicing variant of AIMP2 derives a several signaling cascades, which are crucial for cancer proliferation. Detecting an exon-2 depleted splicing variant (AIMP2-DX2) is an issue of growing importance in cancer therapy. This study suggests the evidence for interrelation between the AIMP2-DX2 and cancer development. We analyzed AIMP2 and AIMP2-DX2 gene expression and their ratio on 7 commercial cancer cell lines and Multiple myeloma patient derived 536MM cell line by RT-PCR and targeted RNA sequencing. Extended this profile, the distribution of AIMP2-DX2/AIMP2 ratio and AIMP2-related major cancer pathways were analyzed using the samples in the ICGC/TCGA database. Over 23 cancer types, 753 samples were used in WTS analysis. In the DEG set analysis, 10 pre-defined major cancer pathways were analyzed among 16 cancer types. Some cancer types, especially acute myeloid leukemia (AML) showed most significant association with AIMP2-DX2 in terms of cancer signaling pathways. We focused on clinical implications of AIMP2-DX2/AIMP2 ratio in the ICGC/TCGA database. 19 AML samples were used, Overall survival (OS) showed that patients with AIMP2-DX2/AIMP2 ratio higher than Q1 shows poor OS and Most of the genes including MEK1/2, ERK, MNK1/2 in this pathway had positive association with AIMP2-DX2/AIMP2 ratio. In colon carcinoma and hepatocellular carcinoma, OS curves had a tendency in a similar way to AML. For the clinical validation of the prognostic value of AIMP2-DX2, 51 AML patients were included in this analysis. The correlation between AIMP2-DX2 expression and survival outcomes was investigated in clinical validation cohort of AML. The AIMP2-DX2-positive group had significantly inferior OS rate and had worse RFS compare to AIMP2-DX2-negative group. Our sequential data shows that the AIMP2-DX2/AIMP2 expression and their ratio can possibly be an indicator to measure malignancy of various cancer types. Citation Format: Dong Chan Kim, Ryul Kim, Daeyoon Kim, Hyojin Song, Dong-Yeop Shin, Inho Kim, Kwang-Sung Ahn, Nam Hoon Kwon, Sunghoon Kim, Sung-Soo Yoon, Youngil Koh. The implications of splicing variant of AIMP2 lacking exon 2 among various cancer types: An analysis of the ICGC/TCGA database and clinical validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 384. doi:10.1158/1538-7445.AM2017-384

Abstract 3186: Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR

Sensitive detection of internal tandem duplication (ITD) mutation of FLT3 is very important in acute myeloid leukemia. To increase detection sensitivity of FLT3-ITD, we developed new detection algorithm using next generation sequencing (NGS) data. We validated results using nested polymerase chain reaction (PCR) methods. We compared results of NGS data, nested PCR and conventional PCR methods. First, using whole exome sequencing data of 83 AML patients, we applied calling algorithm for FLT3-ITD. Briefly, to detect ITDs with NGS data, the reads are aligned to a reference sequence (UCSC hg19), with BWA which is a read aligner allowing soft-clipping. Some reads can be an indication of the occurrence of ITD and BWA aligns those reads as soft-clipped. Second, we deigned two types of primer for Nested PCR. The first primer was targeted wildly for between exon14 and exon15 of FLT3 gene. Nested PCR primer was deigned to target previously reported regions which are frequently occurred ITD mutation. PCR reactions of two steps were performed using the PCR primers sequentially. In these 83 patients, FLT3-ITD was positive only in 7 patients when tested by conventional PCR methods. When NGS detection method was applied, this resulted in positive FLT3-ITD in 11 patients (11/83, 13%). When validation was performed using nested PCR, FLT3-ITD was confirmed in all of 11 patients. Nested PCR detected additional 4 patient positive for FLT3-ITD in this population. For 68 patients, FLT3-ITD was negative by both NGS and nested PCR method. Overall, NGS method improved sensitivity of FLT3-ITD detection by 57% in this population. And the concordance rate of NGS method and nested PCR was 95.2% (79/83). Then we investigated clinical significance of sensitive FLT3-ITD detection. For this, we performed nested PCR and conventional PCR at the same time in 238 AML patients to detect FLT3-ITD. Positive rate for FLT3-ITD was 20% (48/238) and 10% (24/238) by nested PCR and conventional PCR respectively. When survival analysis was performed, among patients with negative FLT3-ITD result by conventional PCR, patients who showed positive for FLT3-ITD by nested PCR had shorter overall survival compared to those who showed negative for FLT3-ITD by nested PCR. (p = 0.03). This implies that sensitive FLT3-ITD detection using nested PCR is clinically meaningful. Diagnosis of FLT3-ITD is very important genetic factor, leading a therapeutic direction for AML patient. Here we report that we have developed alternative more sensitive detection methods for FLT3-ITD based on nested PCR and NGS. Sensitive detection of FLT3-ITD was clinically meaningful, suggesting that these methods should be incorporated in a future clinical practice. Also, we want to note that, NGS method is capable of quantifying FLT3-ITD size and amount in AML patients. Citation Format: Daeyoon Kim, Yoojin Hong, Youngil Koh, Sung-Soo Yoon, Choong-Hyun Sun, Kwang-Sung Ahn, Seungmook Lee, Hongseok Yun, Suyeon Lee. Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3186.

Abstract 4492: Investigation of the germline mutational landscape of GBA in general cancer patients and its role in the tumorigenesis

Introduction Gaucher disease (GD) is an autosomal recessive disease resulting from defective enzymatic activity of acid β-glucosidase (β-glucocerebrosidase). Approximately 400 point mutations in GBA, located in 1q21, have been described and genotype-phenotype correlation was revealed. Previous studies have shown the association of type 1 GD and development of various types of cancers. However, the mutational landscape of GBA and its clinical implication in general cancer patients have not been systematically explored. Methods We used publicly available data obtained from ICGC and TCGA data portal (sftp://dccsftp.nci.nih.gov) for the analysis of single nucleotide variants (SNVs) in GBA in general cancer patients. GenBank reference sequence NM_001005741 was used for SNP identification. Results A total of 2124 cancer patients comprising 29 cancer types were included. Mutations with allele frequency lower than 0.3 or coverage depth lower than 15 were filtered out from analysis. Overall, 7% (151 out of 2124) were GBA mutation carriers, including 1 nonsense (involving exon 1) and 153 missense mutations, including 5 homozygous alternate. The most frequently detected SNVs included rs2230288 (c.1093G>A:p.E365K, 3 homozygous alternate), rs75548401 (c.1223C>T:p.T408M), rs143187997 (c.58A>G:p.I20V, 1 homozygous alternate), rs150466109 (c.38A>G:p.K13R, 1 homozygous alternate), rs76763715 (c.A1226G:p.N409S), and rs421016 (c.1448T>C, p.L483P), accounting for 1.88%, 1.55%, 1.27%, 1.04%, 0.85%, and 0.28% of the entire cancer population, respectively. We compared our data with 1000 Genomes data of 2504 normal subjects (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502), which revealed that the prevalence of rs2230288, rs75548401, and rs76763715 carriers was significantly higher in the cancer group than 1000 Genomes population (p = 0.01, Conclusion Although overall prevalence of germline GBA mutation carriers was not significantly different between cancer patients and general population collected from 1000 Genomes data, SNVs in some specific positions showed statistically significant difference in prevalence between 2 groups, implying association between specific mutational loci of GBA and cancer-developing mechanism, even in heterozygous carriers. Citation Format: Junghoon Shin, Daeyoon Kim, Youngil Koh, Sungsoo Yoon. Investigation of the germline mutational landscape of GBA in general cancer patients and its role in the tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4492.

Abstract 5225: CDK11B, PTPRN2 and WDPCP were frequently duplicated genes in refractory/relapsed normal karyotype AML patients: Identifying structural variations using whole genome sequencing

Background According to the NCCN guideline, the prognosis of acute myeloid leukemia with normal karyotype (NK-AML) is classified based on the mutational status of NPM1, FLT3-ITD or CEBPA. However, patients with NK-AML without the above mutations, who are in intermediate risk, still show various clinical outcomes. Unfortunately, refractory/relapsed patients with intermediate risk NK-AML cannot be predicted before cytotoxic chemotherapy, yet. With the advent of next generation sequencing technology, we tried to reveal prognostic molecular marker in intermediate risk NK-AML, especially focusing on large structural variations (SVs). Methods A total of nine patients with intermediate risk NK-AML patients were included. Whole genome sequencing (WGS) was performed using a pair of tumor and germline DNA of the patients. Leukemic blasts were obtained from bone marrow aspiration specimens of each patient at the time of diagnosis. As a germline control, epithelial cells were collected from saliva of each patient at the time of complete remission. Then, genomic DNA was massively sequenced using HiSeq X10 system (Illumina Inc., San Diego, CA, USA). Possible SVs including inversion, duplication and translocation were identified with BreakDancer (Washington University School of Medicine, St. Louis, MO, USA), Pindel (The Wellcome Trust Sanger Institute, Cambridge, UK) and Delly (European Molecular Biology Laboratory, Heidelberg, Germany). Possible SVs were considered when they were recurrently identified from the three pipelines. Results This cohort consisted of 7 males and 2 females (median age = 55.6 years old). None of them had secondary AML. Eight patients were relapsed after complete remission and the other patient was refractory to 1st line induction chemotherapy. Through WGS, 25 inversions, 67 duplications and 10 translocations were identified in nine patients. Among them, 4 inversions, 13 duplications and 2 translocations were recurrently mutated. Especially, CDK11B, PTPRN2 and WDPCP were frequently duplicated more than 40% of the patients. Conclusions In this study, we performed WGS to investigate SVs of refractory/relapsed NK-AML patients with intermediate risk. Among various mutations, duplications of CDK11B, PTPRN2 and WDPCP were highly prevalent, so they could be a possible prognostic biomarker in NK-AML with intermediate risk. Further functional validation is required in order to clarify their roles. Citation Format: Jeonghwan Youk, Sunghoon Cho, Daeyoon Kim, Youngil Koh, Inho Kim, Murim Choi, Sung-Soo Yoon. CDK11B, PTPRN2 and WDPCP were frequently duplicated genes in refractory/relapsed normal karyotype AML patients: Identifying structural variations using whole genome sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5225.

Abstract 84: A discovery on splicing variant patterns of leucine-tRNA synthetase gene based on targeted RNA sequencing

A leucine-tRNA synthetase, which belongs to the class I aminoacyl-tRNA synthetase family, is encoded by LARS gene. LARS gene functions as an intracellular leucine sensor for mTORC1, mammalian target of rapamycin C1 signaling pathway. This cytosolic gene has a catalytic role of ligating leucine amino acid to its cognate tRNA, in ATP-dependent manner. Accordingly, the protein synthesis regulation, protein translation, cell size, and autophagy are known to be reported by this gene. Therefore, a comprehensive study on LARS gene is necessary for the sake of advanced drug research and precise personalized cancer therapy, in relation to mTOR pathway, which is the root cause of cancer genesis. We discovered the differently expressed splicing patterns in 16 transcripts in LARS gene. Alternative splicing variants of LARS gene were sequenced of 12 cancer cell lines using targeted RNA sequencing to verify their possibilities as a drug target; 12 cancer cell lines are consist of 8 hematologic cell lines and 4 solid cancer cell lines. Sequencing data of LARS gene is aligned by TopHat, and consequently transcript assembly and coverage percentage calculation are processed by Cufflinks, afterwards. Our data, observed from the integrated analysis of these values, shows outstanding features on three transcripts: known protein coding transcript LARS-001 (ENST00000394434, known processed transcript LARS-013 (ENST00000511505), and transcript LARS-015 (ENST00000508709), which is known to retain intron sequences. First of all, the coverage percentage value of transcript LARS-001 in a multiple myeloma cell line, KMS-12-BM, was substantially different from the other cell lines. Secondly, an exceptionally high coverage percent value of the transcript LARS-013 in HL-60, an acute promyelocytic leukemia cell line, was shown. Lastly, the transcript LARS-015 showed a considerably high coverage percent value in SK-MES-1, which is a lung squamous cell carcinoma cell line. Three distinct features of LARS transcripts, transcript LARS-001 in KMS-12-BM, transcript LARS-013 in HL-60, and transcript LARS-015 in SK-MES-1, were characterized. Interestingly, transcript LARS-001 is transcribed with no exon skipping and is only highly expressed in KMS-12-BM. On the other hand, the serial exon skipping from exon 14 to exon 26 in major transcript LARS-201 has shown commonly in 12 cell lines. This implies that LARS gene has unique patterns of alternative splicing variants, which can be consider of taking a significant role in carcinogenesis across diverse cancer types. Hence, additional analysis on exploring the biologically functional correlation between LARS gene and cancer, including cohort validation is to be demonstrated in the future research. Citation Format: HyoJin Song, Daeyoon Kim, Hyejoo Park, Hyun Sub Cheong, Sunghoon Kim, Youngil Koh, Sung-Soo Yoon. A discovery on splicing variant patterns of leucine-tRNA synthetase gene based on targeted RNA sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 84.