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Aims Familial hypercholesterolemia (FH) is currently a worldwide health issue. Understanding the characteristics of patients is important for proper diagnosis and treatment. This study aimed to analyze the phenotypic and genetic features, including threshold cholesterol levels, of Korean patients with FH. Methods A total of 296 patients enrolled in the Korean FH registry were included, according to the following criteria: low-density lipoprotein-cholesterol (LDL-C) ＞190 mg/dL with tendon xanthoma or family history compatible with FH, or LDL-C ＞225 mg/dL. DNA sequences of three FH-associated genes were obtained using whole-exome or target exome sequencing. Threshold cholesterol levels for differentiating patients with FH/pathogenic variant (PV) carriers and predictors of PVs were identified. Results Of the 296 patients, 104 had PVs and showed more obvious clinical findings, including higher cholesterol levels. PV rates ranged from 30% to 64% when patients were categorized by possible or definite type according to the Simon Broome criteria. Frequent PV types included missense variants and copy number variations (CNVs), while the most frequent location of PVs was p.P685L in LDLR. The threshold LDL-C levels for patient differentiation and PV prediction were 177 and 225 mg/dL, respectively. Younger age, tendon xanthoma, and higher LDL-C levels were identified as independent predictors of PVs, while traditional cardiovascular risk factors were predictors of coronary artery disease. Conclusions Korean patients with FH had variable PV rates depending on diagnostic criteria and distinctive PV locations. The reported threshold LDL-C levels pave the way for efficient patient care in this population.

When patients with cancer present with tumors that have migrated from elsewhere in the body, it is difficult for clinicians to identify where the cancer originated. DNA methylation profiling is a promising test to help identify where the cancer originated because it reflects cell of origin and is compatible with formalin-fixed, paraffin-embedded tissues. Because next-generation sequencing has already been implemented in clinical laboratories, the authors developed a targeted bisulfite sequencing panel that could predict the tissue of origin using genomic DNA extracted from formalin-fixed, paraffin-embedded tissues. The authors found that a hybrid capture-based targeted bisulfite sequencing panel is a useful method for predicting the tissue of origin in patients with cancer of unknown primary origin in clinical practice.

Prognostic role of tumor mutation burden (TMB) stratified by microsatellite instability (MSI) status.

Purpose:Recent sequencing studies revealed that a subset of colorectal cancer harbors a significantly higher number of somatic mutations. These hypermutated tumors show distinct clinicopathologic features. However, the prognostic impact of the hypermutated tumors is not clearly established.Experimental Design:We analyzed tumor mutation burden (TMB) from targeted next-generation sequencing data of 40 major genes in 516 patients with colorectal cancer. TMB was defined as total number of nonsynonymous mutations per tumor. Cutoff value for TMB-high was chosen by which best discriminated relapse-free survival (RFS) using the Contal and O'Quigley method.Results:In the TCGA data, mutation count of the selected 40 genes reflected the whole exome mutation burden (Pearson correlation = 0.873, P < 0.001). In our patient cohort, 8 or more mutations in the 40 genes was defined as TMB-high, which best discriminated RFS. A total of 55 patients (10.7%) had TMB-high. TMB-high tumors were more frequently found in a proximal location (63.6%) and had a higher proportion of N0 disease (30.9%) and MSI-H (49.1%) compared with TMB-low. Most importantly, TMB-high was associated with better 5-year RFS compared with TMB-low (96.3% vs. 79.8%, P = 0.005). Although there was significant overlap between TMB-high and MSI-H, MSI-H status was not significantly associated with RFS. Multivariate analysis revealed TMB-high as an independent positive prognostic factor for RFS [adjusted HR, 0.16 (95% confidence interval, 0.04–0.66), P = 0.011].Conclusions:TMB-high is associated with better prognosis in patients with colorectal cancer treated with curative surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy.

Tumor mutation burden in MSI-H patients

Circulating tumor DNA (ctDNA) may reveal dynamic tumor status during therapy. We conducted serial ctDNA analysis to investigate potential association with clinical outcome in metastatic colorectal cancer (mCRC) patients receiving chemotherapy. Tissue KRAS/NRAS wild-type mCRC patients were enrolled and treated with first-line cetuximab-containing chemotherapy. ctDNA isolated from plasma were analyzed by next generation sequencing (NGS) with 16 targeted gene panel. Among 93 patients, 84 (90.3%) had at least 1 somatic mutation in baseline ctDNA samples (average 2.74). Five patients with KRAS or NRAS hotspot mutation in the ctDNA showed significantly worse progression-free survival (PFS) (p = 0.029). Changes in average variant allele frequency (VAF) in ctDNA showed significant correlation with tumor size change at the time of first response evaluation (p = 0.020) and progressive disease (PD) (p = 0.042). Patients whose average VAF decreased below cutoff (p p = 0.018). At the time of PD, 54 new mutations including KRAS and MAP2K1 emerged in ctDNA. ctDNA sequencing can provide mutation profile that could better reflect tumor mutation status and predict treatment outcome.

Purpose: Recent sequencing studies revealed that a subset of colorectal cancer harbors a significantly higher number of somatic mutations. These hypermutated tumors show distinct clinicopathologic features. However, the prognostic impact of the hypermutated tumors is not clearly established. Experimental Design: We analyzed tumor mutation burden (TMB) from targeted next-generation sequencing data of 40 major genes in 516 patients with colorectal cancer. TMB was defined as total number of nonsynonymous mutations per tumor. Cutoff value for TMB-high was chosen by which best discriminated relapse-free survival (RFS) using the Contal and O'Quigley method. Results: In the TCGA data, mutation count of the selected 40 genes reflected the whole exome mutation burden (Pearson correlation = 0.873, P < 0.001). In our patient cohort, 8 or more mutations in the 40 genes was defined as TMB-high, which best discriminated RFS. A total of 55 patients (10.7%) had TMB-high. TMB-high tumors were more frequently found in a proximal location (63.6%) and had a higher proportion of N0 disease (30.9%) and MSI-H (49.1%) compared with TMB-low. Most importantly, TMB-high was associated with better 5-year RFS compared with TMB-low (96.3% vs. 79.8%, P = 0.005). Although there was significant overlap between TMB-high and MSI-H, MSI-H status was not significantly associated with RFS. Multivariate analysis revealed TMB-high as an independent positive prognostic factor for RFS [adjusted HR, 0.16 (95% confidence interval, 0.04–0.66), P = 0.011]. Conclusions: TMB-high is associated with better prognosis in patients with colorectal cancer treated with curative surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy.

BACKGROUND Colorectal cancer (CRC) develops through the alteration of several critical pathways. This study was aimed at evaluating the influence of critical pathways on survival outcomes for patients with CRC. METHODS Targeted next-generation sequencing of 40 genes included in the 5 critical pathways of CRC (WNT, P53, RTK-RAS, phosphatidylinositol-4,5-bisphosphate 3-kinase [PI3K], and transforming growth factor β [TGF-β]) was performed for 516 patients with stage III or high-risk stage II CRC treated with surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy. The associations between critical pathway mutations and relapse-free survival (RFS) and overall survival were analyzed. The associations were further analyzed according to the tumor location. RESULTS The mutation rates for the WNT, P53, RTK-RAS, PI3K, and TGF-β pathways were 84.5%, 69.0%, 60.7%, 30.0%, and 28.9%, respectively. A mutation in the PI3K pathway was associated with longer RFS (adjusted hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.36-0.99), whereas a mutation in the RTK-RAS pathway was associated with shorter RFS (adjusted HR, 1.60; 95% CI, 1.01-2.52). Proximal tumors showed a higher mutation rate than distal tumors, and the mutation profile was different according to the tumor location. The mutation rates of Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), and B-Raf proto-oncogene serine/threonine kinase (BRAF) were higher in proximal tumors, and the mutation rates of adenomatous polyposis coli (APC), tumor protein 53 (TP53), and neuroblastoma RAS viral oncogene homolog (NRAS) were higher in distal tumors. The better RFS with the PI3K pathway mutation was significant only for proximal tumors, and the worse RFS with the RTK-RAS pathway mutation was significant only for distal tumors. CONCLUSIONS A PI3K pathway mutation was associated with better RFS for CRC patients treated with adjuvant chemotherapy, and an RTK-RAS pathway mutation was associated with worse RFS. The significance of the prognostic impact differed according to the tumor location. Cancer 2017;123:3513-23. © 2017 American Cancer Society.

In the present study, we developed a computational method and panel markers to assess microsatellite instability (MSI) using a targeted next-generation sequencing (NGS) platform and compared the performance of our computational method, mSILICO, with that of mSINGS to detect MSI in CRCs. We evaluated 13 CRC cell lines, 84 fresh and 119 formalin-fixed CRC tissues (including 61 MSI-high CRCs and 155 microsatellite-stable CRCs) and tested the classification performance of the two methods on 23, 230, and 3,154 microsatellite markers. For the fresh tissue and cell line samples, mSILICO showed a sensitivity of 100% and a specificity of 100%, regardless of the number of panel markers, whereas for the formalin-fixed tissue samples, mSILICO exhibited a sensitivity of up to 100% and a specificity of up to 100% with three differently sized panels ranging from 23 to 3154. These results were similar to those of mSINGS. With the application of mSILICO, the small panel of 23 markers had a sensitivity of ≥95% and a specificity of 100% in cell lines/fresh tissues and formalin-fixed tissues of CRC. In conclusion, we developed a new computational method and microsatellite marker panels for the determination of MSI that does not require paired normal tissues. A small panel could be integrated into the targeted NGS panel for the concurrent analysis of single nucleotide variations and MSI.

Phenotypic differences in drug responses have been associated with known pharmacogenomic loci, but many remain to be characterized. Therefore, we developed next-generation sequencing (NGS) panels to enable broad and unbiased inspection of genes that are involved in pharmacokinetics (PKs) and pharmacodynamics (PDs). These panels feature repetitively optimized probes to capture up to 114 PK/PD-related genes with high coverage (99.6%) and accuracy (99.9%). Sequencing of a Korean cohort (n = 376) with the panels enabled profiling of actionable variants as well as rare variants of unknown functional consequences. Notably, variants that occurred at low frequency were enriched with likely protein-damaging variants and previously unreported variants. Furthermore, in vitro evaluation of four pharmacogenes, including cytochrome P450 2C19 (CYP2C19), confirmed that many of these rare variants have considerable functional impact. The present study suggests that targeted NGS panels are readily applicable platforms to facilitate comprehensive profiling of pharmacogenes, including common but also rare variants that warrant screening for personalized medicine.

Table S1. Mutation rate of 40 genes included in the study. Table S2. Mutation rate of critical pathways according to histology. Table S3. Survival after recurrence according to each gene mutation. (Genes with mutation rate over 5%). Table S4. Detailed profile of TGF-β pathway mutation. (DOCX 60 kb)

Scalable and cost-effective production of error-free DNA is critical to meet the increased demand for such DNA in the field of biological science. Methods based on ‘Dial-out PCR’ have enabled the high-throughput error-free DNA synthesis from a microarray-synthesized DNA pool by labeling with retrieval PCR tags, and retrieving error-free DNA of which the sequence is identified via next generation sequencing (NGS). However, most of the retrieved products contain byproducts due to background amplification of redundantly labeled DNAs. Here, we present a highly selective retrieval method of desired DNA from a pool of millions of DNA clones from NGS platforms. Our strategy is based on replicating entire sequence-verified DNA molecules from NGS plates to obtain population-controlled DNA pool. Using the NGS-replica pool, we could perform improved and selective retrieval of desired DNA from the replicated DNA pool compared to other dial-out PCR based methods. To evaluate the method, we tested this strategy by using 454, Illumina, and Ion Torrent platforms for producing NGS-replica pool. As a result, we observed a highly selective retrieval yield of over 95%. We anticipate that applications based on this method will enable the preparation of high-fidelity sequenced DNA from heterogeneous collections of DNA molecules.

Colorectal cancer (CRC) develops through the alteration of several critical pathways. This study was aimed at evaluating the influence of critical pathways on survival outcomes for patients with CRC.Targeted next-generation sequencing of 40 genes included in the 5 critical pathways of CRC (WNT, P53, RTK-RAS, phosphatidylinositol-4,5-bisphosphate 3-kinase [PI3K], and transforming growth factor β [TGF-β]) was performed for 516 patients with stage III or high-risk stage II CRC treated with surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy. The associations between critical pathway mutations and relapse-free survival (RFS) and overall survival were analyzed. The associations were further analyzed according to the tumor location.The mutation rates for the WNT, P53, RTK-RAS, PI3K, and TGF-β pathways were 84.5%, 69.0%, 60.7%, 30.0%, and 28.9%, respectively. A mutation in the PI3K pathway was associated with longer RFS (adjusted hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.36-0.99), whereas a mutation in the RTK-RAS pathway was associated with shorter RFS (adjusted HR, 1.60; 95% CI, 1.01-2.52). Proximal tumors showed a higher mutation rate than distal tumors, and the mutation profile was different according to the tumor location. The mutation rates of Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), and B-Raf proto-oncogene serine/threonine kinase (BRAF) were higher in proximal tumors, and the mutation rates of adenomatous polyposis coli (APC), tumor protein 53 (TP53), and neuroblastoma RAS viral oncogene homolog (NRAS) were higher in distal tumors. The better RFS with the PI3K pathway mutation was significant only for proximal tumors, and the worse RFS with the RTK-RAS pathway mutation was significant only for distal tumors.A PI3K pathway mutation was associated with better RFS for CRC patients treated with adjuvant chemotherapy, and an RTK-RAS pathway mutation was associated with worse RFS. The significance of the prognostic impact differed according to the tumor location. Cancer 2017;123:3513-23. © 2017 American Cancer Society.

We developed a highly scalable ‘shotgun’ DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.

The fundamental problem for low-cost gene synthesis is errors that occur during the synthetic process. To address this problem, we developed a practical method that exploits the fact that the predominant errors are deletions. In this method, a simple fluorescence-based readout was used to distinguish error-free synthetic DNA molecules. To do this, we constructed vectors that contained multiple cloning sites and GFP. In the vectors, the GFP gene is designed to be out-of-frame, but insertion of an in-framed synthetic DNA construct into the appropriate cloning site will lead to fluorescent cell colonies. We successfully used this method to synthesize five genes and improved the bp per error from 629 to 6552 by selecting green fluorescent colonies.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder that is characterized by insulin resistance and hyperglycemia. Leptin inhibits the glucose-stimulated insulin secretion, and leptin receptors are present on beta cells as well as on fat cells, thus enabling leptin to modulate both insulin secretion and action. Therefore, leptin (LEP) and leptin receptor (LEPR) genes could play a role in the regulation of glucose and insulin after an oral glucose load. For the association study of LEP and LEPR with T2DM and metabolic traits, 752 women from Seoul National University Hospital (SNUH data) and 532 women from the Korean Health and Genome Study (KHGS data) were selected. Using the SNUH data, we identified that LEP-632G>A and +4998A>C polymorphisms were marginally associated with T2DM, LEP+4950G>A was significantly associated with several metabolic traits, and LEPR+5193G>A, +7187A>C, +27265G>A, +35861T>C, and +52289A>G showed strongly significant association with body mass index (BMI). We observed reproducibility of these results using the KHGS data; LEP+4950G>A and +4998A>C were significantly associated with systolic blood pressure and low-density lipoprotein cholesterol level, respectively. In conclusion, we observed that several polymorphisms in LEPR that had previous reports of association with BMI were significantly replicated in our samples and newly found that some variations of LEP were associated with T2DM and metabolic traits.

Background Although genomic data of CRC continue to accumulate, only limited information on prognostic role of the alterations have been reported. We have analyzed the prognostic impact of the key mutations in CRC using next generation sequencing technology. Methods We selected 40 genes from 5 critical pathways (WNT, TGF-B, PI3K, RTK-RAS and P53) of CRC based on TCGA data. Homogenous population of CRC patients were used to investigate the prognostic implication: 188 stage III or high-risk stage II CRC patients treated with curative surgery followed by adjuvant 5-fluorouracil/ leucovorin/oxaliplatin (FOLFOX) chemotherapy. Archival tissue from Seoul National University Hospital Tumor Bank was used. The target (size 109kb) enrichment process was proceeded base on in-solution hybridization with biotinylated probes. The captured library was amplified and sequenced using Hiseq 2500 (Illumina, USA). Sequencing data was aligned to GRCh37 and variant call and somatic analysis processes was performed by VarScan2 and were annotated with ANNOVAR. Using somatic nonsynonymous mutation data, we selected mutant gene signature which is associated with prognosis. We used binary collapsing method in a forward stepwise selection method to combine mutated genes. Test statistics were obtained from Cox-proportional hazard model. Two-sided p-values of less than 0.05 were considered statistically significant. Results Among a total of 188 patients, 63 had tumor in proximal location and 125 had tumor in distal location. Tumor stage was high-risk stage II in 21 patients and III in 167 patients. Average coverage of the total samples was 417X (414X tumor and 420X normal mucosa). Mutation frequencies were similar with the TCGA data except for NRAS and DKK2, having lower frequency in the present study. During a median follow-up duration of 58 months, 45 relapse and 23 death events have occurred. Alteration in individual gene or pathway did not have prognostic significance in terms of disease free survival (DFS) and overall survival (OS). By using binary collapsing method, we identified a 4 gene signature comprising ACVR1B, ERBB2, LRP5, and KRAS. 89 patients (47.3%) had 1 or more mutation in these 4 genes. 4 gene mutant group had worse DFS and OS compared with 4 gene wild type group. 3-year DFS and 5-year OS was 72.8% and 82.5% in mutant group compared with 88.7% and 96.0% in wild type group (p-values 0.004 and 0.001, respectively). Multivariate analysis revealed 4 gene signature as an independent negative prognostic factor of DFS and OS (adjusted hazard ratios 2.48 and 4.26, respectively). Conclusion Mutations in the 4 genes (ACVR1B, ERBB2, LRP5, and KRAS) were associated with poor prognosis in CRC patients treated with surgery followed by adjuvant FOLFOX chemotherapy. Validation study in an independent cohort is currently underway. Citation Format: Dae-Won Lee, Yongjun Cha, Sae-Won Han, Si-Hyun Lee, Hwang-Phill Kim, Jaemyun Lyu, Hyojun Han, Hyoki Kim, Hoon Jang, Duhee Bang, Iksoo Huh, Taesung Park, Jeong Mo Bae, Jae-Kyung Won, Seung-Yong Jeong, Kyu Joo Park, Gyeong Hoon Kang, Tae-You Kim. Mutation signature associated with colorectal cancer prognosis identified by targeted next-generation sequencing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A72.