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Traditional tissue-based assessments of genomic alterations in castration-resistant prostate cancer (CRPC) can be challenging. To evaluate the real-world clinical utility of liquid biopsies for the evaluation of genomic alterations in CRPC, we preemptively collected available plasma samples and archival tissue samples from patients that were being treated for clinically confirmed CRPC. The cell-free DNA (cfDNA) and tumor tissue DNA were analyzed using the AlphaLiquid®100-HRR panel. Plasma samples from a total of 87 patients were included in this study. Somatic mutations from cfDNA were detected in 78 (89.7%) patients, regardless of the presence of overt metastasis or concomitant treatment given at the time of plasma sample collection. Twenty-three patients were found to have known deleterious somatic or germline mutations in HRR genes from their cfDNA. Archival tissue samples from 33 (37.9%) patients were available for comparative analysis. Tissue sequencing was able to yield an NGS result in only 51.5% of the tissue samples. The general sensitivity of cfDNA for detecting somatic mutations in tissues was 71.8%, but important somatic/germline mutations in HRR genes were found to have a higher concordance (100%). Liquid biopsies can be a reasonable substitute for tissue biopsies in CRPC patients when evaluating genomic alterations.

We present an in-depth single cell atlas of in vitro multi-culture systems on human primary airway epithelium derived from normal and diseased lungs of 27 individual donors. Our large-scale single cell profiling identifies new cell states and differentiation trajectories of rare airway epithelial cell types in human distal lungs. By integrating single cell datasets of human lung tissues, we discover immune primed subsets enriched in lungs and organoids derived from patients with chronic respiratory disease. To demonstrate the full potential of our platform, we further illustrate transcriptomic responses to various respiratory virus infections in in vitro airway models. Our work constitutes a single-cell roadmap for the cellular and molecular characteristics of human primary lung cells in vitro and their relevance to human tissues in vivo.

Ciliary movements within the human airway are essential for maintaining a clean lung environment. Motile cilia have a characteristic ciliary beat frequency (CBF). However, CBF measurement with current video microscopic techniques can be error-prone due to the use of the single-point Fourier transformation, which is often biased for ciliary measurements. Herein, we describe a new video microscopy technique that harnesses a metric of motion-contrast imaging and image correlation for CBF analysis. It can provide objective and selective CBF measurements for individual motile cilia and generate CBF maps for the imaged area. The measurement performance of our methodology was validated with in vitro human airway organoid models that simulated an actual human airway epithelium. The CBF determined for the region of interest (ROI) was equal to that obtained with manual counting. The signal redundancy problem of conventional methods was not observed. Moreover, the obtained CBF measurements were robust to optical focal shifts, and exhibited spatial heterogeneity and temperature dependence. This technique can be used to evaluate ciliary movement in respiratory tracts and determine whether it is non-synchronous or aperiodic in patients. Therefore, our observations suggest that the proposed method can be clinically adapted as a screening tool to diagnose ciliopathies.

Background:Assessment of genomic alterations in castration-resistant prostate cancer (CRPC) patients has become an important process for treatment decisions, but traditional tissue-based evaluation can be challenging. Methods:To evaluate the clinical utility of liquid biopsy for the evaluation of genomic alterations in CRPC, we have prospectively collected plasma samples from patients being treated for clinically confirmed CRPC. The cell-free DNA (cfDNA) extracted from the plasma samples were analyzed using AlphaLiquid®100-HRR panel. Available archival tumor tissue samples were also collected and analyzed using the same panel. Results:Plasma samples from a total of 87 patients were included in this study. Somatic mutations from cfDNA were detected in 78 (89.7%) of all 87 CRPC patients. Somatic mutations were detectable regardless of the presence of overt metastasis, or concomitant treatment given at the time of plasma sample collection. 23 patients were found to have known deleterious mutations in HRR genes from their cfDNA. 11 patients were additionally found to have possibly pathogenic mutations calculated by pathogenicity prediction algorithm. Archival tissue samples were available in 33 (37.9%) of the patients, 31 from prostate and 2 from metastatic bone lesions, collected a median of 2 years before the blood sample collection. Tissue sequencing was able to yield an NGS result in only 51.5% of the tissue samples. The general sensitivity of cfDNA for detecting somatic mutations discovered from tissues was 71.8%, but important somatic/germline mutations in HRR genes were found in higher concordance (100%). Conclusions:Liquid biopsy can be a reasonable substitute for tissue biopsy in CRPC patients for evaluating genomic alterations.

Colorful clones in the blood Stem cells in regenerating tissues such as the blood can acquire mutations that enable a growth advantage, increasing the chance of developing cancer. It is unclear how such diverse mutations promote clonal fitness. Avagyan et al . generated a platform in zebrafish to label clones with unique hues while inducing mutations in genes implicated in human blood disorders. Mutations in some genes caused clones to expand over time, resulting in clonal dominance. Progenitors in the dominant clone expressed anti-inflammatory factors to resist the inflammatory environment produced by their own mature progeny, leading to a self-perpetuating cycle promoting clonal fitness. Targeting these resistance pathways may be used to abate clonal hematopoiesis and prevent its associated pathology. —BAP

Although prime editors are a powerful tool for genome editing, which can generate various types of mutations such as nucleotide substitutions, insertions, and deletions in the genome without double-strand breaks or donor DNA, the conventional prime editors are still limited to their target scopes because of the PAM preference of the Streptococcus pyogenes Cas9 (spCas9) protein. Here, we describe the engineered prime editors to expand the range of their target sites using various PAM-flexible Cas9 variants. Using the engineered prime editors, we could successfully generate more than 50 types of mutations with up to 51.7% prime-editing activity in HEK293T cells. In addition, we successfully introduced the BRAF V600E mutation, which could not be induced by conventional prime editors. These variants of prime editors will broaden the applicability of CRISPR-based prime editing technologies in biological research.

Introduction Blood eosinophils are a predictive marker for the use of inhaled corticosteroids (ICS). However, there is concern over whether a single measure of blood eosinophils is sufficient for outlining a treatment plan. Here, we evaluated the association between variability in blood eosinophils and the effects of ICS in stable COPD cohorts. Methods COPD patients in the Korean COPD Subtype Study and the Seoul National University Airway Registry from 2011 to 2018 were analyzed. Based on blood eosinophils at baseline and at 1-year follow-up, the patients were classified into four groups with 250/μL as a cutoff value: consistently high (CH), consistently low (CL), variably increasing (VI), and variably decreasing (VD). We compared rates of acute exacerbations (AEs) according to ICS use in each group after calibration of severity using propensity score matching. Results Of 2,221 COPD patients, 618 were analyzed and a total of 125 (20%), 355 (57%), 63 (10%), and 75 (12%) patients were classified into the CH, CL, VI, and VD groups, respectively. After calibration, we found that ICS users tended to have a lower AE rate in the CH group (RR 0.41, 95% CI 0.21-0.74) and VI group (RR 0.45, 95% CI 0.22-0.88), but not in the CL group (RR 1.42, 95% CI 1.08-1.89) and VD group (RR 1.71, 95% CI 1.00-2.96). Conclusion More than one-fifth of patients had an inconsistent blood eosinophil level after the 1-year follow-up, and the AE-COPD rate according to ICS differed based on variability in eosinophils. Regular follow-up of blood eosinophils is required for COPD patients.

Background Although single-cell RNA sequencing of xenograft samples has been widely used, no comprehensive bioinformatics pipeline is available for human and mouse mixed single-cell analyses. Considering the numerous homologous genes across the human and mouse genomes, misalignment errors should be evaluated, and a new algorithm is required. We assessed the extents and effects of misalignment errors and exonic multi-mapping events when using human and mouse combined reference data and developed a new bioinformatics pipeline with expression-based species deconvolution to minimize errors. We also evaluated false-positive signals presumed to originate from ambient RNA of the other species and address the importance to computationally remove them. Result Error when using combined reference account for an average of 0.78% of total reads, but such reads were concentrated to few genes that were greatly affected. Human and mouse mixed single-cell data, analyzed using our pipeline, clustered well with unmixed data and showed higher k-nearest-neighbor batch effect test and Local Inverse Simpson’s Index scores than those derived from Cell Ranger (10 × Genomics). We also applied our pipeline to multispecies multisample single-cell library containing breast cancer xenograft tissue and successfully identified all samples using genomic array and expression. Moreover, diverse cell types in the tumor microenvironment were well captured. Conclusion We present our bioinformatics pipeline for mixed human and mouse single-cell data, which can also be applied to pooled libraries to obtain cost-effective single-cell data. We also address misalignment, multi-mapping error, and ambient RNA as a major consideration points when analyzing multispecies single-cell data.

Background: Since complete blood count (CBC) can be easily and repeatedly measured at the usual clinical setting, the various CBC biomarkers including eosinophil count, neutrophil-lymphocyte ratio, platelet-lymphocyte ratio, and eosinophil-basophil ratio have been studied for COPD. Recently, the roles of eosinophil-platelet interactions were emphasized from in vitro or animal model studies. Here, we extensively evaluated the impacts of CBC profiles including eosinophil-platelet interaction on clinical outcomes of stable COPD in Korean cohorts. Methods: The COPD patients enrolled in Korean COPD Subtype Study (KOCOSS) or Seoul National University Airway Registry between 2011 and 2018 were analyzed. Based on the CBC profiles at the time of enrollment, their association with the rate of acute exacerbation (AE) within one year and the annual decline rate of forced expiratory volume in 1 second (FEV1) were evaluated by using negative binomial regression model and linear mixed-effects model. Results: Among 2,221 COPD patients, 1,202 patients were analyzed. Multivariate analysis showed that baseline FEV1 is the most significant factor for both AE rate and FEV1 decline rate. High EBR is associated with high moderate-to-severe AE rates, and both EBR and NLR were related to severe AE rates in addition to FEV1 and the history of AE events. The patients with low eosinophil counts tended faster FEV1 decline, however, platelet counts or eosinophil-platelet interaction terms were not significant factors for outcomes. Conclusion: EBR and NLR are independent prognostic markers for the moderate-to-severe AE and FEV1 decline rate of stable COPD, while eosinophil-platelet interaction had a limited role in predicting clinical outcomes.

Background Nontuberculous mycobacterium (NTM) species are ubiquitous microorganisms. NTM pulmonary disease (NTM-PD) is thought to be caused not by human-to-human transmission but by independent environmental acquisition. However, recent studies using next-generation sequencing (NGS) have reported trans-continental spread of Mycobacterium abscessus among patients with cystic fibrosis. Results We investigated NTM genomes through NGS to examine transmission patterns in three pairs of co-habiting patients with NTM-PD who were suspected of patient-to-patient transmission. Three pairs of patients with NTM-PD co-habiting for at least 15 years were enrolled: a mother and a daughter with M. avium-PD, a couple with M. intracellulare-PD, and a second couple, one of whom was infected with M. intracellulare and the other of whom was infected with M. abscessus. Whole genome sequencing was performed using patients’ NTM isolates as well as environmental specimens. Genetic distances were estimated based on single nucleotide polymorphisms (SNPs). By comparison with the genetic distances among 78 publicly available NTM genomes, NTM isolates derived from the two pairs of patients infected with the same NTM species were not closely related to each other. In phylogenetic analysis, the NTM isolates from patients with M. avium-PD clustered with isolates from different environmental sources. Conclusions In conclusion, considering the genetic distances between NTM strains, the likelihood of patient-to-patient transmission in pairs of co-habiting NTM-PD patients without overt immune deficiency is minimal.

Background : Nontuberculous mycobacterium (NTM) species are ubiquitous microorganisms. NTM pulmonary disease (NTM-PD) is caused not by human-to-human transmission but by independent environmental acquisition. However, recent studies using next-generation sequencing (NGS) have reported trans-continental spread of Mycobacterium abscessus among patients with cystic fibrosis. Results : We investigated NTM genomes through NGS to examine transmission patterns in three pairs of co-habiting NTM-PD patients who were suspected of patient-to-patient transmission. Three pairs of patients with NTM-PD co-habiting for at least 15 years were enrolled: a mother and a daughter with M. avium PD, a couple with M. intracellulare PD, and a second couple, one of whom was infected with M. intracellulare PD and the other of whom was infected with M. abscessus subsp. massiliense PD. Whole genome sequencing was performed using NTM colonies isolated from patients and environmental specimens. Genetic distances were estimated based on single nucleotide polymorphisms (SNPs) in the NTM genomes. Comparing SNPs in the consensus regions, the minimum pairwise SNP distances of NTM isolates derived from the two pairs of patients infected with the same NTM species were over 10,000. In phylogenetic analysis, the NTM isolates from patients with M. avium PD clustered with isolates from different environmental sources. Conclusions : In conclusion, considering the genetic distances between NTM strains, the likelihood of patient-to-patient transmission in pairs of co-habiting NTM-PD patients without overt immune deficiency is minimal.

OBJECTIVE Remarkable difference in cellular activity was found between early and late subpassaged embryonic stem cell (ESCs) lines, which can be created by subtle changes in cell manipulation protocol. This study subsequently examined whether post-thaw subculture of early subpassaged ESC lines could further affect the activity of the ESCs. METHODS Fresh (as a control treatment) or cryopreserved F1 hybrid (B6CBAF1) early ESC lines (C57BL/6xCBA) of the 4 (P4) or the 19 passage (P19) were subcultured once, twice or six times under the same condition. The post-thaw survival of the ESCs was monitored after the post-treatment subculture and the ability of cell proliferation, reactive oxygen species (ROS) generation, apoptosis and mitochondrial ATP synthesis was subsequently examined. RESULTS Regardless of the subculture number, P19 ESCs showed better (p

Jung-Ki Yoon,1,2 Arthur E Frankel,3 Lynn G Feun,4 Suhendan Ekmekcioglu,1 Kevin B Kim11Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA;2Hwasung Public Health Center, Hwasung, South Korea, 3Scott and White Cancer Research Institute, Temple, TX, USA;4Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, USAAbstract: Despite recent development of promising immunotherapeutic and targeted drugs, prognosis in patients with advanced melanoma remains poor, and a cure for this disease remains elusive in most patients. The success of melanoma therapy depends on a better understanding of the biology of melanoma and development of drugs that effectively target the relevant genes or proteins essential for tumor cell survival. Melanoma cells frequently lack argininosuccinate synthetase, an essential enzyme for arginine synthesis, and as a result they become dependent on the availability of exogenous arginine. Accordingly, a therapeutic approach involving depletion of available arginine has been shown to be effective in preclinical studies. Early clinical studies have demonstrated sufficient antitumor activity to give rise to cautious optimism. In this article, the rationale for arginine deprivation therapy is discussed. Additionally, various strategies for depleting arginine are discussed and the preclinical and clinical investigations of arginine deprivation therapy in melanoma are reviewed.Keywords: arginine deprivation, argininosuccinate synthetase, melanoma

Utilizing the full power of next-generation sequencing often requires the ability to perform large-scale multiplex enrichment of many specific genomic loci in multiple samples. Several technologies have been recently developed but await substantial improvements. We report the 10,000-fold improvement of a previously developed padlock-based approach, and apply the assay to identifying genetic variations in hypermutable CpG regions across human chromosome 21. From approximately 3 million reads derived from a single Illumina Genome Analyzer lane, approximately 94% (approximately 50,500) target sites can be observed with at least one read. The uniformity of coverage was also greatly improved; up to 93% and 57% of all targets fell within a 100- and 10-fold coverage range, respectively. Alleles at >400,000 target base positions were determined across six subjects and examined for single nucleotide polymorphisms (SNPs), and the concordance with independently obtained genotypes was 98.4%-100%. We detected >500 SNPs not currently in dbSNP, 362 of which were in targeted CpG locations. Transitions in CpG sites were at least 13.7 times more abundant than non-CpG transitions. Fractions of polymorphic CpG sites are lower in CpG-rich regions and show higher correlation with human-chimpanzee divergence within CpG versus non-CpG sites. This is consistent with the hypothesis that methylation rate heterogeneity along chromosomes contributes to mutation rate variation in humans. Our success suggests that targeted CpG resequencing is an efficient way to identify common and rare genetic variations. In addition, the significantly improved padlock capture technology can be readily applied to other projects that require multiplex sample preparation.

The protein enzyme ribonuclease A (RNaseA) cleaves RNA with catalytic perfection, although with little sequence specificity, by a divalent metal ion (M(2+))-independent mechanism in which a pair of imidazoles provides general acid and base catalysis, while a cationic amine provides electrostatic stabilization of the transition state. Synthetic imitation of this remarkable organo-catalyst ("RNaseA mimicry") has been a longstanding goal in biomimetic chemistry. The 9(25)-11 DNAzyme contains synthetically modified nucleotides presenting both imidazole and cationic amine side chains, and catalyzes RNA cleavage with turnover in the absence of M(2+) similarly to RNaseA. Nevertheless, the catalytic roles, if any, of the "protein-like" functional groups have not been defined, and hence the question remains whether 9(25)-11 engages any of these functionalities to mimic aspects of the mechanism of RNaseA. To address this question, we report a mechanistic investigation of 9(25)-11 catalysis wherein we have employed a variety of experiments, such as DNAzyme functional group deletion, mechanism-based affinity labeling, and bridging and nonbridging phosphorothioate substitution of the scissile phosphate. Several striking parallels exist between the results presented here for 9(25)-11 and the results of analogous experiments applied previously to RNaseA. Specifically, our results implicate two particular imidazoles in general acid and base catalysis and suggest that a specific cationic amine stabilizes the transition state via diastereoselective interaction with the scissile phosphate. Overall, 9(25)-11 appears to meet the minimal criteria of an RNaseA mimic; this demonstrates how added synthetic functionality can expand the mechanistic repertoire available to a synthetic DNA-based catalyst.

Interpreting epistatic interactions is crucial for understanding evolutionary dynamics of complex genetic systems and unveiling structure and function of genetic pathways. Although high resolution mapping of en masse variant libraries renders molecular biologists to address genotype-phenotype relationships, long-read sequencing technology remains indispensable to assess functional relationship between mutations that lie far apart. Here, we introduce JigsawSeq for multiplexed sequence identification of pooled gene variant libraries by combining a codon-based molecular barcoding strategy and de novo assembly of short-read data. We first validate JigsawSeq on small sub-pools and observed high precision and recall at various experimental settings. With extensive simulations, we then apply JigsawSeq to large-scale gene variant libraries to show that our method can be reliably scaled using next-generation sequencing. JigsawSeq may serve as a rapid screening tool for functional genomics and offer the opportunity to explore evolutionary trajectories of protein variants., This paper described a new and efficient method for de novo assembly of multiple DNA sequence information from mutagenized clone libraries. Using codon-barcoded libraries and calling the method JigsawSeq, the authors overcome limitations of short-read sequencing assembly from next-generation sequencing.

Molecular inversion probe (MIP)-based capture is a scalable and effective target-enrichment technology that can use synthetic single-stranded oligonucleotides as probes. Unlike the straightforward use of synthetic oligonucleotides for low-throughput target capture, high-throughput MIP capture has required laborious protocols to generate thousands of single-stranded probes from DNA microarray because of multiple enzymatic steps, gel purifications and extensive PCR amplifications. Here, we developed a simple and efficient microarray-based MIP preparation protocol using only one enzyme with double-stranded probes and improved target capture yields by designing probes with overlapping targets and unique barcodes. To test our strategy, we produced 11 510 microarray-based duplex MIPs (microDuMIPs) and captured 3554 exons of 228 genes in a HapMap genomic DNA sample (NA12878). Under our protocol, capture performance and precision of calling were compatible to conventional MIP capture methods, yet overlapping targets and unique barcodes allowed us to precisely genotype with as little as 50 ng of input genomic DNA without library preparation. microDuMIP method is simpler and cheaper, allowing broader applications and accurate target sequencing with a scalable number of targets.

Background The extent to which metastatic tumors further evolve by accumulating additional mutations is unclear and has yet to be addressed extensively using next-generation sequencing of high-grade serous ovarian cancer. Methods Eleven spatially separated tumor samples from the primary tumor and associated metastatic sites and two normal samples were obtained from a Stage IIIC ovarian cancer patient during cytoreductive surgery prior to chemotherapy. Whole exome sequencing and copy number analysis were performed. Omental exomes were sequenced with a high depth of coverage to thoroughly explore the variants in metastatic lesions. Somatic mutations were further validated by ultra-deep targeted sequencing to sort out false positives and false negatives. Based on the somatic mutations and copy number variation profiles, a phylogenetic tree was generated to explore the evolutionary relationship among tumor samples. Results Only 6% of the somatic mutations were present in every sample of a given case with TP53 as the only known mutant gene consistently present in all samples. Two non-spatial clusters of primary tumors (cluster P1 and P2), and a cluster of metastatic regions (cluster M) were identified. The patterns of mutations indicate that cluster P1 and P2 diverged in the early phase of tumorigenesis, and that metastatic cluster M originated from the common ancestral clone of cluster P1 with few somatic mutations and copy number variations. Conclusions Although a high level of intratumor heterogeneity was evident in high-grade serous ovarian cancer, our results suggest that transcoelomic metastasis arises with little accumulation of somatic mutations and copy number alterations in this patient. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1077-4) contains supplementary material, which is available to authorized users.

Ultrasonography of a cat with diarrhoea and vomiting revealed a multi-layered, discrete linear structure within the large intestine with retention of the intestinal layers which could potentially be confused with an intestinal intussusception. The structure was ultimately expelled from the large intestine during defecation, and confirmed as a fibrinonecrotic cast. The origin of the fibrinonecrotic cast was assumed to be an intestinal pseudo-membrane formed in enteritis caused by immune suppression due to the panleukopenia virus. To our knowledge, this is the first ultrasonographic description of a fibrinonecrotic cast and spontaneous passage of the colonic cast in the veterinary field.

Editing Expectations Although genetic information is stored in DNA, and faithfully copied into RNA, the cell can make the odd (and occasionally vitally important) change to the meaning of code during a process known as RNA editing. Thirteen edited genes are known in the nonrepetitive portion of the human genome, but the overall prevalence of RNA editing is unclear. Li et al. (p. 1210 ), used an unbiased genome-wide approach to identify 239 sites (in 207 target genes), with stringent criteria for editing. The sites identified included 10 of the 13 known edited genes. Fourteen out of 18 randomly chosen sites were validated by sequencing, and these putatively edited genes were enriched for synapse, cell trafficking, and membrane functions. Furthermore, lowering the search stringency suggested that many more human genes may be edited at lower frequencies.

Background: Lung adenocarcinoma is a highly heterogeneous disease with various etiologies, prognoses, and responses to therapy. Although genome-scale characterization of lung adenocarcinoma has been performed, somatic mutation analysis of never-smoker EGFR/KRAS/ALK-negative lung adenocarcinoma has not been comprehensively conducted. Methods: Patients with lung adenocarcinoma were eligible if they were never smoker without EGFR/KRAS mutation and ALK rearrangement. Whole exome sequencing was performed using the Illumina HiSeq 2,000 platform. Candidate loci identified in whole exome sequencing were validated using hybridization capture method and Sanger sequencing. Results: We present an analysis of whole exome sequencing data from 16 EGFR/KRAS/ALK-negative lung adenocarcinomas with additional 54 tumors in two independent cohort sets. We identified 27 genes potentially implicated in the pathogenesis of lung adenocarcinoma. In addition to targetable PI3K/mTOR signaling (TSC1, PIK3CA, AKT2) and receptor tyrosine kinase (RTK) signaling (ERBB4) genes, some of the genes including SETD2 and PBRM1 (chromatin remodeling), CHEK2 and CDC27 (cell cycle), CUL3 and SOD2 (oxidative stress), and CSMD3 and TFG (immune response) have not been previously highlighted in lung adenocarcinomas. In total (N=70), TP53 is the most frequently altered gene (11%), followed by SETD2 (6%), CSMD3 (6%), ERBB2 (6%), and CDH10 (4%). In pathway analysis, majority of altered genes were involved in cell cycle/DNA repair (P Conclusions: Genomic makeup of EGFR/KRAS/ALK-negative adenocarcinomas in never-smokers was remarkably diverse. Impairment in cell cycle/DNA repair may be involved in tumorigenesis and therapeutic targets in lung adenocarcinoma. Note: This abstract was not presented at the meeting. Citation Format: Han Sang Kim, Jin Woo Ahn, Jung-Ki Yoon, Soo Min Han, Hoon Jang, Sungho Eun, Hyo Sup Shim, Hyun Jung Kim, Dae Joon Kim, Jin Gu Lee, Chang Young Lee, Mi Kyung Bae, Kyung Young Chung, Eun Young Kim, Ji Ye Jung, Se Kyu Kim, Joon Chang, Hye Ryun Kim, Joo Hang Kim, Ji Hyun Lee, Duhee Bang, Byoung Chul Cho. Identification of somatic mutations in EGFR/KRAS/ALK-negative lung adenocarcinoma from never smokers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4158. doi:10.1158/1538-7445.AM2014-4158

Background: The extent to which metastatic tumors further evolve by accumulating additional mutations is unclear and has yet to be systematically addressed using next-generation sequencing in high-grade serous ovarian cancer. Methods: During cytoreductive surgery prior to chemotherapy, 11 spatially separated tumor samples from primary tumor and associated metastatic sites with 2 normal samples were obtained. Whole exome sequencing and copy number analysis were performed and multiplex PCR followed by ultra-deep resequencing was used for validation. Especially, omental samples were sequenced with high depth of coverage (∼200x).Phylogenetic tree was built to explore the evolutionary relationship between samples. Results: Only 6% of all somatic mutations were present in every sample of a given case with TP53 gene as the only known mutant gene consistently present in all samples. Two non-spatial clusters of primary samples (cluster 1 and 2), and a cluster of metastatic samples (cluster M) were identified. The patterns of mutations indicate that cluster M was originated from cluster 1, and that cluster 2 was diverged from a portion of right fimbriae in cluster 1 at the early phase of tumorigenesis. No significant metastatic sample-specific mutations or copy number changes were detected. Conclusion: Our results showed that high level of intratumor heterogeneity was evident in a high-grade serous ovarian cancer. Moreover, we showed that transcoelomic metastasis arises with little accumulation of somatic mutations or copy number alterations. Citation Format: Jung-Yun Lee, Jung-Ki Yoon, Duhee Bang, Yong-Sang Song. Identification of somatic mutations and copy number alterations in metastatic high-grade ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1547. doi:10.1158/1538-7445.AM2014-1547

Background: Lung adenocarcinoma is a highly heterogeneous disease with various etiologies, prognoses, and responses to therapy. Although genome-scale characterization of lung adenocarcinoma has been performed, a comprehensive somatic mutation analysis of EGFR/KRAS/ALK-negative lung adenocarcinoma in never-smokers has not been conducted. Methods: We analyzed whole exome sequencing data from 16 EGFR/KRAS/ALK-negative lung adenocarcinomas and additional 54 tumors in two expansion cohort sets. Candidate loci were validated by target capture and Sanger sequencing. Gene set analysis was performed using Ingenuity Pathway Analysis. Results: We identified 27 genes potentially implicated in the pathogenesis of lung adenocarcinoma. These included targetable genes involved in PI3K/mTOR signaling (TSC1, PIK3CA, AKT2) and receptor tyrosine kinase signaling (ERBB4) and genes not previously highlighted in lung adenocarcinomas, such as SETD2 and PBRM1 (chromatin remodeling), CHEK2 and CDC27 (cell cycle), CUL3 and SOD2 (oxidative stress), and CSMD3 and TFG (immune response). In the expansion cohort (N = 70), TP53 was the most frequently altered gene (11%), followed by SETD2 (6%), CSMD3 (6%), ERBB2 (6%), and CDH10 (4%). In pathway analysis, the majority of altered genes were involved in cell cycle/DNA repair (P