ABSTRACT
Purpose@#For precision medicine, exploration and monitoring of molecular biomarkers are essential. However, in advanced gastric cancer (GC) with visceral lesions, an invasive procedure cannot be performed repeatedly for the follow-up of molecular biomarkers. @*Materials and Methods@#To verify the clinical implication of serial liquid biopsies targeting circulating tumor DNA (ctDNA) on treatment response, we conducted targeted deep sequencing for serially collected ctDNA of 15 HER2-positive metastatic GC patients treated with anti-PD-1 inhibitor in combination with standard systemic treatment. @*Results@#In the baseline ctDNAs, 14 patients (93%) harbored more than one genetic alteration. A number of mutations in wellknown cancer-related genes, such as KRAS and PIK3CA, were identified. Copy number alterations were identified in eight GCs (53.3%), and amplification of the ERBB2 gene (6/15, 40.0%) was the most recurrent. When we calculated the mean variant allele frequency (VAF) of mutations in each ctDNA as the molecular tumor burden index (mTBI), the mTBI trend was largely consistent with the VAF profiles in both responder and non-responder groups. Notably, in the longitudinal analysis of ctDNA, mTBI provided 2–42 weeks (mean 13.4 weeks) lead time in the detection of disease progression compared to conventional follow-up with CT imaging. @*Conclusion@#Our data indicate that the serial genetic alteration profiling of ctDNA is feasible to predict treatment response in HER2-positive GC patients in a minimally invasive manner. Practically, ctDNA profiles are useful not only for the molecular diagnosis of GC but also for the selection of GC patients with poor prognosis for systemic treatment (ClinicalTrials.gov identifier:NCT02901301).
ABSTRACT
Influenza A virus (IAV) is the most widespread pathogen causing human respiratory infections. Although polymerase chain reaction (PCR)–based methods are currently the mostcommonly used tools for IAV detection, PCR is not ideal for point-of-care testing. In thisstudy, we aimed to develop a more rapid and sensitive method than PCR-based tools todetect IAV using loop-mediated isothermal amplification (LAMP) technology. We designedreverse-transcriptional (RT)–LAMP primers targeting the hemagglutinin gene. RNAs fromreference H1N1 and H3N2 showed specific RT-LAMP signals with the designed primers.We optimized the reaction conditions and developed universal reaction conditions for bothLAMP assays. Under these conditions, the detection limit was 50 copies for both RT-LAMPassays. There was no non-specific signal to 19 non-IAV respiratory viruses, such as influenza B virus, coronaviruses, and respiratory syncytial viruses. Regarding the reaction time, apositive signal was detected within 25 min after starting the reaction. In conclusion, ourRT-LAMP assay has high sensitivity and specificity for the detection of the H1 and H3 subtypes, making it suitable for point-of-care IAV testing.
ABSTRACT
Loss of heterozygosity (LOH) is a genomic aberration. In some cases, LOH can be generated without changing the copy number, which is called copy-neutral LOH (CN-LOH). CN-LOH frequently occurs in various human diseases, including cancer. However, the biological and clinical implications of CN-LOH for human diseases have not been well studied. In this study, we compared the performance of CN-LOH determination using three commonly used tools. For an objective comparison, we analyzed CN-LOH profiles from single-nucleotide polymorphism array data from 10 colon adenocarcinoma patients, which were used as the reference for comparison with the CN-LOHs obtained through whole-exome sequencing (WES) data of the same patients using three different analysis tools (FACETS, Nexus, and Sequenza). The majority of the CN-LOHs identified from the WES data were consistent with the reference data. However, some of the CN-LOHs identified from the WES data were not consistent between the three tools, and the consistency with the reference CN-LOH profile was also different. The Jaccard index of the CN-LOHs using FACETS (0.84 ± 0.29; mean value, 0.73) was significantly higher than that of Nexus (0.55 ± 0.29; mean value, 0.50; p = 0.02) or Sequenza (0 ± 0.41; mean value, 0.34; p = 0.04). FACETS showed the highest area under the curve value. Taken together, of the three CN-LOH analysis tools, FACETS showed the best performance in identifying CN-LOHs from The Cancer Genome Atlas colon adenocarcinoma WES data. Our results will be helpful in exploring the biological or clinical implications of CN-LOH for human diseases.
ABSTRACT
Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.
ABSTRACT
In addition to mutations and copy number alterations, gene fusions are commonly identified in cancers. In thyroid cancer, fusions of important cancer-related genes have been commonly reported; however, extant panels do not cover all clinically important gene fusions. In this study, we aimed to develop a custom RNA-based sequencing panel to identify the key fusions in thyroid cancer. Our ThyChase panel was designed to detect 87 types of gene fusion. As quality control of RNA sequencing, five housekeeping genes were included in this panel. When we applied this panel for the analysis of fusions containing reference RNA (HD796), three expected fusions (EML4-ALK, CCDC6-RET, and TPM3-NTRK1) were successfully identified. We confirmed the fusion breakpoint sequences of the three fusions from HD796 by Sanger sequencing. Regarding the limit of detection, this panel could detect the target fusions from a tumor sample containing a 1% fusion-positive tumor cellular fraction. Taken together, our ThyChase panel would be useful to identify gene fusions in the clinical field.
ABSTRACT
Highly pathogenic avian influenza (HPAI) viruses have caused severe respiratory disease and death in poultry and human beings. Although most of the avian influenza viruses (AIVs) are of low pathogenicity and cause mild infections in birds, some subtypes including hemagglutinin H5 and H7 subtype cause HPAI. Therefore, sensitive and accurate subtyping of AIV is important to prepare and prevent for the spread of HPAI. Next-generation sequencing (NGS) can analyze the full-length sequence information of entire AIV genome at once, so this technology is becoming a more common in detecting AIVs and predicting subtypes. However, an analysis pipeline of NGS-based AIV sequencing data, including AIV subtyping, has not yet been established. Here, in order to support the pre-processing of NGS data and its interpretation, we developed a user-friendly tool, named prediction of avian influenza virus subtype (PAIVS). PAIVS has multiple functions that support the pre-processing of NGS data, reference-guided AIV subtyping, de novo assembly, variant calling and identifying the closest full-length sequences by BLAST, and provide the graphical summary to the end users.
ABSTRACT
Avian influenza (AIV) outbreaks can induce fatal human pulmonary infections in addition to economic losses to the poultry industry. In this study, we aimed to develop a rapid and sensitive point-of-care AIV test using loop-mediated isothermal amplification (LAMP) technology. We designed three sets of reverse transcription LAMP (RT-LAMP) primers targeting the matrix (M) and hemagglutinin (HA) genes of the H5 and H9 subtypes. RT-LAMP targeting the universal M gene was designed to screen for the presence of AIV and RT-LAMP assays targeting H5-HA and H9-HA were designed to discriminate between the H5 and H9 subtypes. All three RT-LAMP assays showed specific amplification results without nonspecific reactions. In terms of sensitivity, the detection limits of our RT-LAMP assays were 100 to 1,000 RNA copies per reaction, which were 10 times more sensitive than the detection limits of the reference reverse‒transcription polymerase chain reaction (RT-PCR) (1,000 to 10,000 RNA copies per reaction). The reaction time of our RT-LAMP assays was less than 30 minutes, which was approximately four times quicker than that of conventional RT-PCR. Altogether, these assays successfully detected the existence of AIV and discriminated between the H5 or H9 subtypes with higher sensitivity and less time than the conventional RT-PCR assay.
ABSTRACT
Highly pathogenic avian influenza (HPAI) viruses have caused severe respiratory disease and death in poultry and human beings. Although most of the avian influenza viruses (AIVs) are of low pathogenicity and cause mild infections in birds, some subtypes including hemagglutinin H5 and H7 subtype cause HPAI. Therefore, sensitive and accurate subtyping of AIV is important to prepare and prevent for the spread of HPAI. Next-generation sequencing (NGS) can analyze the full-length sequence information of entire AIV genome at once, so this technology is becoming a more common in detecting AIVs and predicting subtypes. However, an analysis pipeline of NGS-based AIV sequencing data, including AIV subtyping, has not yet been established. Here, in order to support the pre-processing of NGS data and its interpretation, we developed a user-friendly tool, named prediction of avian influenza virus subtype (PAIVS). PAIVS has multiple functions that support the pre-processing of NGS data, reference-guided AIV subtyping, de novo assembly, variant calling and identifying the closest full-length sequences by BLAST, and provide the graphical summary to the end users.
ABSTRACT
Avian influenza (AIV) outbreaks can induce fatal human pulmonary infections in addition to economic losses to the poultry industry. In this study, we aimed to develop a rapid and sensitive point-of-care AIV test using loop-mediated isothermal amplification (LAMP) technology. We designed three sets of reverse transcription LAMP (RT-LAMP) primers targeting the matrix (M) and hemagglutinin (HA) genes of the H5 and H9 subtypes. RT-LAMP targeting the universal M gene was designed to screen for the presence of AIV and RT-LAMP assays targeting H5-HA and H9-HA were designed to discriminate between the H5 and H9 subtypes. All three RT-LAMP assays showed specific amplification results without nonspecific reactions. In terms of sensitivity, the detection limits of our RT-LAMP assays were 100 to 1,000 RNA copies per reaction, which were 10 times more sensitive than the detection limits of the reference reverse‒transcription polymerase chain reaction (RT-PCR) (1,000 to 10,000 RNA copies per reaction). The reaction time of our RT-LAMP assays was less than 30 minutes, which was approximately four times quicker than that of conventional RT-PCR. Altogether, these assays successfully detected the existence of AIV and discriminated between the H5 or H9 subtypes with higher sensitivity and less time than the conventional RT-PCR assay.
ABSTRACT
PURPOSE: With the emergence of next-generation sequencing (NGS) technology, profiling a wide range of genomic alterations has become a possibility resulting in improved implementation of targeted cancer therapy. In Asian populations, the prevalence and spectrum of clinically actionable genetic alterations has not yet been determined because of a lack of studies examining high-throughput cancer genomic data. MATERIALS AND METHODS: To address this issue, 1,071 tumor samples were collected from five major cancer institutes in Korea and analyzed using targeted NGS at a centralized laboratory. Samples were either fresh frozen or formalin-fixed, paraffin embedded (FFPE) and the quality and yield of extracted genomic DNA was assessed. In order to estimate the effect of sample condition on the quality of sequencing results, tissue preparation method, specimen type (resected or biopsied) and tissue storage time were compared. RESULTS: We detected 7,360 non-synonymous point mutations, 1,164 small insertions and deletions, 3,173 copy number alterations, and 462 structural variants. Fifty-four percent of tumors had one or more clinically relevant genetic mutation. The distribution of actionable variants was variable among different genes. Fresh frozen tissues, surgically resected specimens, and recently obtained specimens generated superior sequencing results over FFPE tissues, biopsied specimens, and tissues with long storage duration. CONCLUSION: In order to overcome, challenges involved in bringing NGS testing into routine clinical use, a centralized laboratory model was designed that could improve the NGS workflows, provide appropriate turnaround times and control costs with goal of enabling precision medicine.
Subject(s)
Humans , Academies and Institutes , Asian People , DNA , Korea , Methods , Paraffin , Point Mutation , Precision Medicine , PrevalenceABSTRACT
Robust identification of genetic alterations is important for the diagnosis and subsequent treatment of tumors. Screening for genetic alterations using tumor tissue samples may lead to biased interpretations because of the heterogeneous nature of the tumor mass. Liquid biopsy has been suggested as an attractive tool for the non-invasive follow-up of cancer treatment outcomes. In this study, we aimed to verify whether the mutations identified in primary tumor tissue samples could be consistently detected in plasma cell–free DNA (cfDNA) by digital polymerase chain reaction (dPCR). We first examined the genetic alteration profiles of three colorectal cancer (CRC) tissue samples by targeted next-generation sequencing (NGS) and identified 11 non-silent amino acid changes across six cancer-related genes (APC, KRAS, TP53, TERT, ARIDIA, and BRCA1). All three samples had KRAS mutations (G12V, G12C, and G13D), which were well-known driver events. Therefore, we examined the KRAS mutations by dPCR. When we examined the three KRAS mutations by dPCR using tumor tissue samples, all of them were consistently detected and the variant allele frequencies (VAFs) of the mutations were almost identical between targeted NGS and dPCR. When we examined the KRAS mutations using the plasma cfDNA of the three CRC patients by dPCR, all three mutations were consistently identified. However, the VAFs were lower (range, 0.166% to 2.638%) than those obtained using the CRC tissue samples. In conclusion, we confirmed that the KRAS mutations identified from CRC tumor tissue samples were consistently detected in the plasma cfDNA of the three CRC patients by dPCR.
ABSTRACT
Robust identification of genetic alterations is important for the diagnosis and subsequent treatment of tumors. Screening for genetic alterations using tumor tissue samples may lead to biased interpretations because of the heterogeneous nature of the tumor mass. Liquid biopsy has been suggested as an attractive tool for the non-invasive follow-up of cancer treatment outcomes. In this study, we aimed to verify whether the mutations identified in primary tumor tissue samples could be consistently detected in plasma cell–free DNA (cfDNA) by digital polymerase chain reaction (dPCR). We first examined the genetic alteration profiles of three colorectal cancer (CRC) tissue samples by targeted next-generation sequencing (NGS) and identified 11 non-silent amino acid changes across six cancer-related genes (APC, KRAS, TP53, TERT, ARIDIA, and BRCA1). All three samples had KRAS mutations (G12V, G12C, and G13D), which were well-known driver events. Therefore, we examined the KRAS mutations by dPCR. When we examined the three KRAS mutations by dPCR using tumor tissue samples, all of them were consistently detected and the variant allele frequencies (VAFs) of the mutations were almost identical between targeted NGS and dPCR. When we examined the KRAS mutations using the plasma cfDNA of the three CRC patients by dPCR, all three mutations were consistently identified. However, the VAFs were lower (range, 0.166% to 2.638%) than those obtained using the CRC tissue samples. In conclusion, we confirmed that the KRAS mutations identified from CRC tumor tissue samples were consistently detected in the plasma cfDNA of the three CRC patients by dPCR.
Subject(s)
Humans , Bias , Biopsy , Colorectal Neoplasms , Diagnosis , DNA , Follow-Up Studies , Gene Frequency , Mass Screening , Plasma , Polymerase Chain ReactionABSTRACT
The acquisition of somatic mutations is the most common event in cancer. Neoantigens expressed from genes with mutations acquired during carcinogenesis can be tumor-specific. Since the immune system recognizes tumor-specific peptides, they are potential targets for personalized neoantigen-based immunotherapy. However, the discovery of druggable neoantigens remains challenging, suggesting that a deeper understanding of the mechanism of neoantigen generation and better strategies to identify them will be required to realize the promise of neoantigen-based immunotherapy. Alternative splicing and RNA editing events are emerging mechanisms leading to neoantigen production. In this review, we outline recent work involving the large-scale screening of neoantigens produced by alternative splicing and RNA editing. We also describe strategies to predict and validate neoantigens from RNA sequencing data.
Subject(s)
Humans , Alternative Splicing , Carcinogenesis , Immune System , Immunotherapy , Mass Screening , Peptides , RNA Editing , RNA , Sequence Analysis, RNAABSTRACT
PURPOSE: Papillary renal cell carcinoma (PRCC) gene, which located in 1q23.1, is recurrently amplified in non-small cell lung cancer (NSCLC). However, it is unknown whether PRCC is overexpressed in primary NSCLCs and whether PRCC overexpression contributes to lung tumorigenesis. In this study, we aimed to identify the profiles of PRCC expression in Korean NSCLC patients and to elucidate the role of PRCC overexpression on lung tumorigenesis. MATERIALS AND METHODS: We performed immunohistochemistry analysis with a tissue array containing 161 primary NSCLCs. Small interfering RNA targeting PRCC (siPRCC) was transfected into two lung cancer cell lines (NCI-H358 and A549), after which tumor growth, migration, and invasion were observed. Expressions of cell proliferation-, cell cycle-, and metastasis-related molecules were examined by Western blot analysis. We also explored the in vivo effect of PRCC silencing. RESULTS: PRCC overexpression was recurrently observed in NSCLCs (95/161, 59%). After siPRCC treatment, tumor cell proliferation, colony formation, and anchorage independent growth were significantly reduced (p < 0.001 for all three effects). Migration and invasiveness were also significantly repressed (p < 0.001 for both effects). Reflecting cell proliferation, cell cycle, and metastasis, the expressions of Ki67, cyclin D1, AKT-1, pAKT, NF-kB p65, vimentin and CXCL-12 were found to be downregulated. Through mouse xenograft analysis, we confirmed that PRCC silencing significantly repressed a xenograft tumor mass in vivo (p < 0.001). CONCLUSION: The present data provide evidence that PRCC overexpression is involved in the tumorigenesis and progression of lung cancer.
Subject(s)
Animals , Humans , Mice , Blotting, Western , Carcinogenesis , Carcinoma, Non-Small-Cell Lung , Carcinoma, Renal Cell , Cell Cycle , Cell Line , Cell Proliferation , Cyclin D1 , Heterografts , Immunohistochemistry , Lung Neoplasms , Lung , Neoplasm Metastasis , NF-kappa B , RNA, Small Interfering , VimentinABSTRACT
PURPOSE: Kawasaki disease (KD) is a mucocutaneous lymph node syndrome. It is mainly seen in young children under the age of five. KD is a multifactorial disorder that includes genetic variants. The present study investigated the association between KD and single nucleotide polymorphisms (SNPs) in the candidate gene early B cell factor 2 (EBF2), which is associated with inflammation markers. MATERIALS AND METHODS: An SNP analysis was performed by whole exon sequencing of the EBF2 gene. Our study comprised a total of 495 subjects (295 KD patients and 200 unrelated normal controls) from a Korean population. Tag SNPs were discovered using the Haploview program. Genotyping of the EBF2 gene was performed with the TaqMan® assay with real-time PCR methods. RESULTS: Polymorphism of rs10866845 showed a significant difference in allele frequency between KD patients and controls (p=0.040). The EBF2 gene polymorphisms were significantly associated with KD on logistic regression analysis. CONCLUSION: EBF2 gene variants can contribute to KD in the Korean population.
Subject(s)
Child , Humans , Exons , Gene Frequency , Inflammation , Logistic Models , Mucocutaneous Lymph Node Syndrome , Polymorphism, Single Nucleotide , Real-Time Polymerase Chain ReactionABSTRACT
Vulvar squamous cell carcinoma (SCC) consists of two different etiologic categories: human papilloma virus (HPV)-associated (HPV (+)) and HPV-non-associated (HPV (−)). There have been no genome-wide studies on the genetic alterations of vulvar SCCs or on the differences between HPV (+) and HPV (−) vulvar SCCs. In this study, we performed whole-exome sequencing and copy number profiling of 6 HPV (+) and 9 HPV (−) vulvar SCCs and found known mutations (TP53, CDKN2A and HRAS) and copy number alterations (CNAs) (7p and 8q gains and 2q loss) in HPV (−) SCCs. In HPV (+), we found novel mutations in PIK3CA, BRCA2 and FBXW7 that had not been reported in vulvar SCCs. HPV (−) SCCs exhibited more mutational loads (numbers of nonsilent mutations and driver mutations) than HPV (+) SCCs, but the CNA loads and mutation signatures between HPV (+) and HPV (−) SCCs did not differ. Of note, 40% and 40% of the 15 vulvar SCCs harbored PIK3CA and FAT1 alterations, respectively. In addition, we found that the SCCs harbored kataegis (a localized hypermutation) in 2 HPV (+) SCCs and copy-neutral losses of heterozygosity in 4 (one HPV (+) and 3 HPV (−)) SCCs. Our data indicate that HPV (+) and HPV (−) vulvar SCCs may have different mutation and CNA profiles but that there are genomic features common to SCCs. Our data provide useful information for both HPV (+) and HPV (−) vulvar SCCs and may aid in the development of clinical treatment strategies.
ABSTRACT
Patient-derived xenograft (PDX) models are useful tools for tumor biology research and testing the efficacy of candidate anticancer drugs targeting the druggable mutations identified in tumor tissue. However, it is still unknown how much of the genetic alterations identified in primary tumors are consistently detected in tumor tissues in the PDX model. In this study, we analyzed the genetic alterations of three primary colorectal cancers (CRCs) and matched xenograft tissues in PDX models using a next-generation sequencing cancer panel. Of the 17 somatic mutations identified from the three CRCs, 14 (82.4%) were consistently identified in both primary and xenograft tumors. The other three mutations identified in the primary tumor were not detected in the xenograft tumor tissue. There was no newly identified mutation in the xenograft tumor tissues. In addition to the somatic mutations, the copy number alteration profiles were also largely consistent between the primary tumor and xenograft tissue. All of these data suggest that the PDX tumor model preserves the majority of the key mutations detected in the primary tumor site. This study provides evidence that the PDX model is useful for testing targeted therapies in the clinical field and research on precision medicine.
Subject(s)
Biology , Colorectal Neoplasms , Heterografts , Precision MedicineABSTRACT
No abstract available.
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No abstract available.
Subject(s)
Cyclooxygenase 2 Inhibitors , Spondylitis, Ankylosing , Escherichia coli , Transcriptome , Iran , Bacterial Toxins , Cellular Senescence , Fibroblasts , KoreaABSTRACT
Ankylosing spondylitis (AS) is a chronic autoinflammatory disease that affects the spine and sacroiliac joints. Regarding its etiology, although HLA-B27 is known to be the strongest genetic factor of AS, much evidence suggests the potential contribution of non-MHC genes to the susceptibility to AS. Most of these non-MHC genes have been discovered in non-Asian populations; however, just some of them have been validated in Koreans. In this study, we aimed to identify additional AS-associated single-nucleotide polymorphism (SNP) candidates by replicating the candidate SNPs in Korean AS patients and healthy controls. For this, we selected three SNPs (rs11249215 in RUNX3, rs6556416 in IL12B, and rs8070463 in TBKBP1), which were previously reported as risk factors of AS but have not been studied in Koreans, and performed genotyping assays using a total of 1138 Korean samples (572 AS patients and 566 healthy controls). Of the three SNP candidates, one SNP in RUNX3 (rs11249215) was significantly associated with the risk of AS (odds ratio, 1.31; 95% confidence interval, 1.02 to 1.68, p = 0.03). These results will be helpful in elucidating the pathogenesis of AS and may be useful for developing AS risk prediction models in Koreans.