Hongseok Yun

Revising a personal genome by comparing and combining data from two different sequencing platforms.

For the robust practice of genomic medicine, sequencing results must be compatible, regardless of the sequencing technologies and algorithms used. Presently, genome sequencing is still an imprecise science and is complicated by differences in the chemistry, coverage, alignment, and variant-calling algorithms. We identified ∼3.33 million single nucleotide variants (SNVs) and ∼3.62 million SNVs in the SJK genome using SOLiD and Illumina data, respectively. Approximately 3 million SNVs were concordant between the two platforms while 68,532 SNVs were discordant; 219,616 SNVs were SOLiD-specific and 516,080 SNVs were Illumina-specific (i.e., platform-specific). Concordant, discordant, and platform-specific SNVs were further analyzed and characterized. Overall, a large portion of heterozygous SNVs that were discordant with genotyping calls of single nucleotide polymorphism chips were highly confident. Approximately 70% of the platform-specific SNVs were located in regions containing repetitive sequences. Such platform-specificity may arise from differences between platforms, with regard to read length (36 bp and 72 bp vs. 50 bp), insert size (∼100–300 bp vs. ∼1–2 kb), sequencing chemistry (sequencing-by-synthesis using single nucleotides vs. ligation-based sequencing using oligomers), and sequencing quality. When data from the two platforms were merged for variant calling, the proportion of callable regions of the reference genome increased to 99.66%, which was 1.43% higher than the average callability of the two platforms, representing ∼40 million bases. In this study, we compared the differences in sequencing results between two sequencing platforms. Approximately 90% of the SNVs were concordant between the two platforms, yet ∼10% of the SNVs were either discordant or platform-specific, indicating that each platform had its own strengths and weaknesses. When data from the two platforms were merged, both the overall callability of the reference genome and the overall accuracy of the SNVs improved, demonstrating the likelihood that a re-sequenced genome can be revised using complementary data.

Integrated genomic analyses identify frequent gene fusion events and VHL inactivation in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. We sequenced nine exomes and transcriptomes, and two genomes of GISTs for integrated analyses. We detected 306 somatic variants in nine GISTs and recurrent protein-altering mutations in 29 genes. Transcriptome sequencing revealed 328 gene fusions, and the most frequently involved fusion events were associated with IGF2 fused to several partner genes including CCND1, FUS, and LASP1. We additionally identified three recurrent read-through fusion transcripts: POLA2-CDC42EP2, C8orf42-FBXO25, and STX16-NPEPL1. Notably, we found intragenic deletions in one of three exons of the VHL gene and increased mRNAs of VEGF, PDGF-β, and IGF-1/2 in 56% of GISTs, suggesting a mechanistic link between VHL inactivation and overexpression of hypoxia-inducible factor target genes in the absence of hypoxia. We also identified copy number gain and increased mRNA expression of AMACR, CRIM1, SKP2, and CACNA1E. Mapping of copy number and gene expression results to the KEGG pathways revealed activation of the JAK-STAT pathway in small intestinal GISTs and the MAPK pathway in wild-type GISTs. These observations will allow us to determine the genetic basis of GISTs and will facilitate further investigation to develop new therapeutic options.

Detection of a Distinctive Genomic Signature in Rhabdoid Glioblastoma, A Rare Disease Entity Identified by Whole Exome Sequencing and Whole Transcriptome Sequencing

We analyzed the genome of a rhabdoid glioblastoma (R-GBM) tumor, a very rare variant of GBM. A surgical specimen of R-GBM from a 20-year-old woman was analyzed using whole exome sequencing (WES), whole transcriptome sequencing (WTS), single nucleotide polymorphism array, and array comparative genomic hybridization. The status of gene expression in R-GBM tissue was compared with that of normal brain tissue and conventional GBM tumor tissue. We identified 23 somatic non-synonymous small nucleotide variants with WES. We identified the BRAF V600E mutation and possible functional changes in the mutated genes, ISL1 and NDRG2. Copy number alteration analysis revealed gains of chromosomes 3, 7, and 9. We found loss of heterozygosity and focal homozygous deletion on 9q21, which includes CDKN2A and CDKN2B. In addition, WTS revealed that CDK6, MET, EZH2, EGFR, and NOTCH1, which are located on chromosomes 7 and 9, were over-expressed, whereas CDKN2A/2B were minimally expressed. Fusion gene analysis showed 14 candidate genes that may be functionally involved in R-GBM, including TWIST2, and UPK3BL. The BRAF V600E mutation, CDKN2A/2B deletion, and EGFR/MET copy number gain were observed. These simultaneous alterations are very rarely found in GBM. Moreover, the NDRG2 mutation was first identified in this study as it has never been reported in GBM. We observed a unique genomic signature in R-GBM compared to conventional GBM, which may provide insight regarding R-GBM as a distinct disease entity among the larger group of GBMs.

Genomic dynamics associated with malignant transformation in IDH1 mutated gliomas

The genomic mechanism responsible for malignant transformation remains an open question for glioma researchers, where differing conclusions have been drawn based on diverse study conditions. Therefore, it is essential to secure direct evidence using longitudinal samples from the same patient. Moreover, malignant transformation of IDH1-mutated gliomas is of potential interest, as its genomic mechanism under influence of oncometabolite remains unclear, and even higher rate of malignant transformation was reported in IDH1-mutated low grade gliomas than in wild-type IDH1 tumors. We have analyzed genomic data using next-generation sequencing technology for longitudinal samples from 3 patients with IDH1-mutated gliomas whose disease had progressed from a low grade to a high grade phenotype. Comprehensive analysis included chromosomal aberrations as well as whole exome and transcriptome sequencing, and the candidate driver genes for malignant transformation were validated with public database. Integrated analysis of genomic dynamics in clonal evolution during the malignant transformation revealed alterations in the machinery regulating gene expression, including the spliceosome complex (U2AF2), transcription factors (TCF12), and chromatin remodelers (ARID1A). Moreover, consequential expression changes implied the activation of genes associated with the restoration of the stemness of cancer cells. The alterations in genetic regulatory mechanisms may be the key factor for the major phenotypic changes in IDH1 mutated gliomas. Despite being limited to a small number of cases, this analysis provides a direct example of the genomic changes responsible for malignant transformation in gliomas.

A scientific treatment approach for acute mast cell leukemia: using a strategy based on next-generation sequencing data

Background Mast cell leukemia (MCL) is the most aggressive form of systemic mastocytosis disorders. Owing to its rarity, neither pathogenesis nor standard treatment is established for this orphan disease. Hence, we tried to treat a patient with MCL based on the exome and transcriptome sequencing results of the patients own DNA and RNA. Methods First, tumor DNA and RNA were extracted from bone marrow at the time of diagnosis. Germline DNA was extracted from the patients saliva 45 days after induction chemotherapy and used as a control. Then, we performed whole-exome sequencing (WES) using the DNA and whole transcriptome sequencing (WTS) using the RNA. Single nucleotide variants (SNVs) were called using MuTect and GATK. Samtools, FusionMap, and Gene Set Enrichment Analysis were utilized to analyze WTS results. Results WES and WTS results revealed mutation in KIT S476I. Fusion analysis was performed using WTS data, which suggested a possible RARα-B2M fusion. When RNA expression analysis was performed using WTS data, upregulation of PIK3/AKT pathway, downstream of KIT and mTOR, was observed. Based on our WES and WTS results, we first administered all-trans retinoic acid, then dasatinib, and finally, an mTOR inhibitor. Conclusion We present a case of orphan disease where we used a targeted approach using WES and WTS data of the patient. Even though our treatment was not successful, use of our approach warrants further validation.

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

Genetic Characteristics of Polycythemia Vera and Essential Thrombocythemia in Korean Patients.

Despite recent advances in the investigation of myeloproliferative neoplasms (MPN), the impact of genetic heterogeneity on its molecular pathogenesis has not been fully elucidated. Thus, in this study, we aim to characterize the genetic complexity in Korean patients with polycythemia vera (PV) and essential thrombocythemia (ET).

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.