Original Clinical Sample Research Articles

Screening biomarkers for spinal cord injury using weighted gene co-expression network analysis and machine learning.

JOURNAL/nrgr/04.03/01300535-202412000-00028/figure1/v/2024-04-08T165401Z/r/image-tiff Immune changes and inflammatory responses have been identified as central events in the pathological process of spinal cord injury. They can greatly affect nerve regeneration and functional recovery. However, there is still limited understanding of the peripheral immune inflammatory response in spinal cord injury. In this study, we obtained microRNA expression profiles from the peripheral blood of patients with spinal cord injury using high-throughput sequencing. We also obtained the mRNA expression profile of spinal cord injury patients from the Gene Expression Omnibus (GEO) database (GSE151371). We identified 54 differentially expressed microRNAs and 1656 differentially expressed genes using bioinformatics approaches. Functional enrichment analysis revealed that various common immune and inflammation-related signaling pathways, such as neutrophil extracellular trap formation pathway, T cell receptor signaling pathway, and nuclear factor-κB signal pathway, were abnormally activated or inhibited in spinal cord injury patient samples. We applied an integrated strategy that combines weighted gene co-expression network analysis, LASSO logistic regression, and SVM-RFE algorithm and identified three biomarkers associated with spinal cord injury: ANO10, BST1, and ZFP36L2. We verified the expression levels and diagnostic performance of these three genes in the original training dataset and clinical samples through the receiver operating characteristic curve. Quantitative polymerase chain reaction results showed that ANO10 and BST1 mRNA levels were increased and ZFP36L2 mRNA was decreased in the peripheral blood of spinal cord injury patients. We also constructed a small RNA-mRNA interaction network using Cytoscape. Additionally, we evaluated the proportion of 22 types of immune cells in the peripheral blood of spinal cord injury patients using the CIBERSORT tool. The proportions of naïve B cells, plasma cells, monocytes, and neutrophils were increased while the proportions of memory B cells, CD8+ T cells, resting natural killer cells, resting dendritic cells, and eosinophils were markedly decreased in spinal cord injury patients increased compared with healthy subjects, and ANO10, BST1 and ZFP26L2 were closely related to the proportion of certain immune cell types. The findings from this study provide new directions for the development of treatment strategies related to immune inflammation in spinal cord injury and suggest that ANO10, BST1, and ZFP36L2 are potential biomarkers for spinal cord injury. The study was registered in the Chinese Clinical Trial Registry (registration No. ChiCTR2200066985, December 12, 2022).

Abstract PO013: Patient-derived xenograft mouse models of hepatoblastoma for a personalized medicine pipeline

Abstract Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Patients with high-risk, treatment refractory, or relapse disease have a survival rate of less than 50%. The development of clinically relevant models of these aggressive tumors will facilitate studies to identify drugs that target these cells.Methods: Fresh, whole primary tumor samples were implanted into the livers of immunocompromised mice. Tumor growth was monitored with MRI and ELISA to measure serum human Alpha-fetoprotein (AFP), which is detectable in the blood of tumor-bearing animals. Tumors were validated with immunohistochemistry (IHC) for HB markers Glypican-3 (GPC3) and Beta-catenin; short tandem repeat (STR) DNA validation; next generation sequencing-based mutation profiling of 124 genes involved in pediatric solid tumors; RNA sequencing (RNA-seq), and single cell RNA-seq (scRNA-seq). Lung metastasis was also detected in models with serial sectioning and H&E staining. Cells derived from tumors were grown in vitro in adherent and spheroid conditions and used for high throughput drug screening of candidate agents. Tumors were serially passaged in animals for further in vivo drug testing of novel targeted agents.Results: Nine patient-derived xenograft (PDX) models were generated that represent low- and high-risk tumors, treatment refractory cases, and relapsed tumors. Passaging of these models showed consistent implantation rates at or above 80% with tumors detectable in 2 to 4 weeks. Eight of nine models secrete human serum AFP. All models mimic gene expression and histological patterns of their primary tumor counterparts as well as identical STR DNA profiles. The models also show gene expression consistent with an HB2/high-risk profile according to the Sumazin HB expression signature. Interestingly, two models represent unique sub-clones of a very aggressive HB relapse with different AFP secretion and transcriptomic expression. scRNA-seq of these two models indicated outgrowth of disparate disease sub-clones. The nine models also demonstrate a range of DNA mutations with three or four mutations per tumor; all variants present in the original clinical samples were conserved in the PDX models. Lung metastasis was evident in six of nine models. Two stable patient-derived cell lines (PDCLs) were developed from models, and these cell lines show expression of HB markers and secrete AFP with growth in culture. Drug screening of adherent and spheroid tumor cells support the efficacy of novel targeted agents and indicate a spectrum of sensitivity to cisplatin, a frontline standard chemotherapy agent. Importantly, the models replicate the chemotherapy responses of the corresponding patients. Additional in vitro and in vivo work showed the efficacy of a histone deacetylase inhibitor, panobinostat.Conclusions: These novel orthotopic PDX models of HB fully recapitulate the primary tumors and represent a platform for clinically relevant drug screening and testing. Citation Format: Sarah E Woodfield, Roma H Patel, Andres F Espinoza, Richard S Whitlock, Jessica Epps, Andrew Badachhape, Samuel R Larson, Rohit K Srivastava, Aayushi P Shah, Saiabhiroop R Govindu, Barry Zorman, Brandon J Mistretta, Kevin E Fisher, Ilavarasi Gandhi, Jacquelyn Reuther, Martin Urbicain, Aryana M Ibarra, Sakuni Rankothgedera, Kimberly R Holloway, Stephen F Sarabia, Andras Heczey, Ketan B Ghaghada, Kalyani R Patel, Dolores Lopez-Terrada, Angshumoy Roy, Preethi H Gunaratne, Pavel Sumazin, Sanjeev A Vasudevan. Patient-derived xenograft mouse models of hepatoblastoma for a personalized medicine pipeline [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO013.

Preliminary study on the diagnostic value of serum-derived exosomal lncRNA in epithelial ovarian cancer

Objective: To investigate the diagnostic value of long noncoding RNA (lncRNA) extracted from serum exosomes in epithelial ovarian cancer (EOC). Methods: (1) Patients with ovarian tumors who were hospitalized in the Affiliated Tumor Hospital of Guangxi Medical University from August 2018 to December 2019, including 35 cases of EOC patients (malignant group) and 20 cases of benign ovarian tumor patients (benign group) were collected; during the same period, 15 healthy women (normal group) who underwent physical examination in the Affiliated Tumor Hospital of Guangxi Medical University were used as controls. Fasting venous blood serum was collected from the above three groups of women, and serum exosomes were isolated and purified using commercial kits. The morphology of exosomal particles was observed with transmission electron microscope, and the particle size distribution of the exosomes was detected by NanoSight technology. The expression of specific proteins cluster of differentiation (CD)63, CD81, and tumor susceptibility gene 101 (TSG101) of exosomes were analyzed by western blot. (2) Four cases of EOC patients and three cases of healthy women were randomly selected. High-throughput sequencing technology was used to analyze the differentially expressed lncRNA in serum exosomes of these four EOC patients and three healthy women, and screen out the significantly differentially expressed lncRNA. The screened lncRNA with different expression levels was verified by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) in these seven original clinical samples, furtherly confirmed and tested with QRT-PCR in larger clinical samples (a total of 70 serum samples). (3) The receiver operating characteristic (ROC) curve of the target lncRNA was drawn and its diagnostic indicators such as sensitivity and specificity were evaluated. By using logistic binary regression model, multi-factor joint diagnostic models were constructed and evaluated. Results: (1) Under transmission electron microscope, clear lipid bilayer structure was observed in serum exosomes, and one side presented a concave hemispheric or cup like structure; the peak diameter of the exosomal particles detected with NanoSight technology was 127.6 nm, and the particles between 30 and 150 nm accounted for 58.9%; western blot confirmed that the obtained (exosomal) particles could detect the expression of the marker proteins CD63, CD81, and TSG101. (2) Analysis of high-throughput sequencing technology showed that compared with the women in the normal sequencing group (3 cases), 425 differentially expressed lncRNAs (including 23 up-regulated and 402 down-regulated) were screened in the serum exosomes of the malignant sequencing group (4 cases). Six types of lncRNA with significantly abnormal expression levels (including FER1L6-AS2, LINC00470, LINC01811, CXXC4-AS1, LINC02343, and LINC02428) were randomly selected for original sample verification, and the results were consistent with the sequencing results. Subsequently, these six lncRNAs were used for larger samples QRT-PCR verification. Compared with the benign and normal groups, the expression of FER1L6-AS2, LINC00470 and LINC01811 in malignant group increased by 1.66 and 1.84-fold, 2.05 and 2.46-fold, 2.94 and 2.35-fold, respectively; the expressions of CXXC4-AS1, LINC02343 and LINC02428 were down-regulated to 29% and 34%, 40% and 46%, 42% and 42%, respectively. For the same lncRNA, there were statistical differences between the malignant group and the benign group, between the malignant group and the normal group (all P<0.05), and there were no statistical differences between the benign group and the normal group (all P>0.05). (3) The results showed that the area under curve (AUC) of these six lncRNAs ranged from 0.722 to 0.805, which had moderate diagnostic efficiency. To use logistic binary regression model to establish multi-indicator joint diagnostic models and establish different joint factor ROC curves. The results showed that the AUC of the joint factor prediction model 1 (composed of FER1L6-AS2 and LINC01811), the joint factor prediction model 2 (composed of CXXC4-AS1, LINC02343, and LINC02428), and the joint factor prediction model 3 (composed of FER1L6-AS2, CXXC4-AS1, LINC02343, and LINC02428) were 0.865, 0.934, and 0.962, respectively. The diagnostic efficacy of the combined factor prediction models was higher than that of the single lncRNA (all P<0.05). Conclusions: High-throughput sequencing technology is an effective method for screening out the different expression levels of lncRNA extracted from serum exosomes. The combined detection of multiple serum exosomal lncRNA indicators has a certain diagnostic efficacy for patients with EOC. Detection of serum exosomal lncRNA indicators will provide new ideas for the diagnosis of EOC.

SARS-CoV-2 genetic variations associated with COVID-19 pathogenicity.

In this study, we performed genome-wide association analyses on SARS-CoV-2 genomes to identify genetic mutations associated with pre-symptomatic/asymptomatic COVID-19 cases. Various potential covariates and confounding factors of COVID-19 severity, including patient age, gender and country, as well as virus phylogenetic relatedness were adjusted for. In total, 3021 full-length genomes of SARS-CoV-2 generated from original clinical samples and whose patient status could be determined conclusively as either ‘pre-symptomatic/asymptomatic’ or ‘symptomatic’ were retrieved from the GISAID database. We found that the mutation 11 083G>T, located in the coding region of non-structural protein 6, is significantly associated with asymptomatic COVID-19. Patient age is positively correlated with symptomatic infection, while gender is not significantly correlated with the development of the disease. We also found that the effects of the mutation, patient age and gender do not vary significantly among countries, although each country appears to have varying baseline chances of COVID-19 symptom development.

Grief-related beliefs in shidu parents with and without prolonged grief disorder: Psychometric properties of a Chinese version of the Typical Beliefs Questionnaire.

The Typical Beliefs Questionnaire (TBQ) assesses common grief-related cognitions, which demonstrated satisfactory psychometric properties in a treatment-seeking sample with prolonged grief disorder (PGD). Chinese shidu parents (bereaved parents over the age of 49 who have lost their only child) are at a high risk of PGD. The current study aimed to examine psychometric properties of the Chinese version of the TBQ (TBQ-C) in a community sample of shidu parents with and without PGD, to compare this to the original validation clinical sample in the United States bereaved of any close relationship and to consider its use as a clinical tool. We examined the rate of positive endorsement, factor structure, internal consistency and validity of the TBQ-C in 310 community-based shidu parents (including 102 who met the criteria for PGD). Results showed that the rate of positive endorsement for each item ranged from 7.2% to 48.6% among non-PGD participants and from 20.6% to 92.2% among PGD participants. Confirmatory factor analysis indicated that the original five-factor structure fit both the non-PGD and PGD shidu parents. The TBQ-C showed acceptable internal consistency and satisfactory convergent and concurrent validity in both groups. It had good discriminant validity and can be helpful in distinguishing shidu parents with and without PGD. The TBQ-C can be used to investigate common grief-related beliefs that may be problematic for both shidu parents with and without PGD.

Abstract 2997: Novel orthotopic patient-derived xenograft mouse models of hepatoblastoma that replicate tumor heterogeneity, chemoresistance, and refractory disease

Abstract Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Patients with high-risk, treatment refractory, or relapse disease have a survival rate of less than 50%. The development of clinically relevant models of these aggressive tumors will facilitate studies to identify drugs that target these cells. Methods: Fresh, whole primary tumor samples were implanted into the livers of immunocompromised mice. Tumor growth was monitored with MRI and ELISA to measure serum human Alpha-fetoprotein (AFP), which is detectable in the blood of tumor-bearing animals. Tumors were validated with immunohistochemistry (IHC) for HB markers Glypican-3 (GPC3) and Beta-catenin; short tandem repeat (STR) DNA validation; next generation sequencing-based mutation profiling of 124 genes involved in pediatric solid tumors; and RNA sequencing (RNA-seq). Cells derived from tumors were grown in vitro and used for high throughput drug screening of candidate agents. Tumors were serially passaged in animals for further in vivo drug testing of novel targeted agents. Results: Nine patient-derived xenograft (PDX) models were generated that represent low- and high-risk tumors, treatment refractory cases, and relapsed tumors. Passaging of these models showed consistent implantation rates at or above 80% with tumors detectable in 2 to 4 weeks. Eight of nine models secrete human serum AFP. All models mimic gene expression and histological patterns of their primary tumor counterparts as well as identical STR DNA profiles. The models also show gene expression consistent with an HB2/high-risk profile according to the Sumazin HB expression signature. Interestingly, two models represent unique sub-clones of a very aggressive HB relapse with different AFP secretion and transcriptomic expression. The nine models also demonstrate a range of DNA mutations with three or four mutations per tumor; all variants present in the original clinical samples were conserved in the PDX models. Drug screening of tumor cells support the efficacy of novel targeted agents and indicate that these tumors are resistant to frontline standard chemotherapy, cisplatin and doxorubicin. More extensive in vivo testing showed the efficacy of a dual MDM2/MDM4 inhibitor (ALRN-6924), a cyclin dependent kinase inhibitor (dinaciclib), and a histone deacetylase inhibitor (panobinostat), and use of an orthotopic animal model also revealed drug toxicities associated with compromised liver function. Conclusions: These novel orthotopic PDX models of HB fully recapitulate the primary tumors and represent a platform for clinically relevant drug screening and testing. Further studies of the sub-clones of disease that grow in the animals will yield information about particularly aggressive HBs. Citation Format: Sarah Elizabeth Woodfield, Roma H. Patel, Samuel R. Larson, Andrew Badachhape, Rohit K. Srivastava, Aayushi P. Shah, Brandon Mistretta, Barry Zorman, Kevin Fisher, Ilavarasi Gandhi, Jacquelyn Reuther, Richard S. Whitlock, Aryana M. Ibarra, Sakuni Rankothgedera, Kimberly R. Holloway, Stephen F. Sarabia, Martin Urbicain, Andras Heczey, Dolores Lopez-Terrada, Angshumoy Roy, Preethi Gunaratne, Pavel Sumazin, Sanjeev A. Vasudevan. Novel orthotopic patient-derived xenograft mouse models of hepatoblastoma that replicate tumor heterogeneity, chemoresistance, and refractory disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2997.

Cell-based Culture Informs Infectivity and Safe De-Isolation Assessments in Patients with Coronavirus Disease 2019.

BackgroundThe detection of SARS-CoV-2 RNA by real-time polymerase chain reaction (PCR) in respiratory samples collected from persons recovered from COVID-19 does not necessarily indicate shedding of infective virions. By contrast, the isolation of SARS-CoV-2 using cell-based culture likely indicates infectivity, but there are limited data on the correlation between SARS-CoV-2 culture and PCR.MethodsOne hundred and ninety-five patients with varying severity of COVID-19 were tested (outpatients [n=178]), inpatients [n=12] and critically unwell patients admitted to the intensive care unit [ICU; n=5]). SARS-CoV-2 PCR positive samples were cultured in Vero C1008 cells and inspected daily for cytopathic effect (CPE). SARS-CoV-2-induced CPE was confirmed by PCR of culture supernatant. Where no CPE was observed, PCR was performed on day four to confirm absence of virus replication. Cycle threshold (Ct) of the day four PCR (Ctculture) and the PCR of the original clinical sample (Ctsample) were compared, and positive cultures were defined where Ctsample - Ctculture was ≥3.FindingsOf 234 samples collected, 228 (97%) were from the upper respiratory tract. SARS-CoV-2 was only successfully isolated from samples with Ctsample ≤32, including in 28/181 (15%), 19/42 (45%) and 9/11 samples (82%) collected from outpatients, inpatients, and ICU patients, respectively. The mean duration from symptom onset to culture positivity was 4.5 days (range 0-18). SARS-CoV-2 was significantly more likely to be isolated from samples collected from inpatients (p<0∙001) and ICU patients (p<0∙0001) compared with outpatients respectively, and in samples with lower Ctsample.ConclusionSARS-CoV-2 culture may be used as a surrogate marker for infectivity and inform de-isolation protocols.

Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks

BackgroundToxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain.MethodsSelected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes.ResultsAs a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms.ConclusionsThe present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity.

Limited Intrahost Diversity and Background Evolution Accompany 40 Years of Canine Parvovirus Host Adaptation and Spread.

Canine parvovirus (CPV) is a highly successful pathogen that has sustained pandemic circulation in dogs for more than 40 years. Here, integrating full-genome and deep-sequencing analyses, structural information, and in vitro experimentation, we describe the macro- and microscale features that accompany CPV's evolutionary success. Despite 40 years of viral evolution, all CPV variants are more than ∼99% identical in nucleotide sequence, with only a limited number (<40) of substitutions becoming fixed or widespread during this time. Notably, most substitutions in the major capsid protein (VP2) gene are nonsynonymous, altering amino acid residues that fall within, or adjacent to, the overlapping receptor footprint or antigenic regions, suggesting that natural selection has channeled much of CPV evolution. Among the limited number of variable sites, CPV genomes exhibit complex patterns of variation that include parallel evolution, reversion, and recombination, compromising phylogenetic inference. At the intrahost level, deep sequencing of viral DNA in original clinical samples from dogs and other host species sampled between 1978 and 2018 revealed few subconsensus single nucleotide variants (SNVs) above ∼0.5%, and experimental passages demonstrate that substantial preexisting genetic variation is not necessarily required for rapid host receptor-driven adaptation. Together, these findings suggest that although CPV is capable of rapid host adaptation, a relatively low mutation rate, pleiotropy, and/or a lack of selective challenges since its initial emergence have inhibited the long-term accumulation of genetic diversity. Hence, continuously high levels of inter- and intrahost diversity are not necessarily required for virus host adaptation.IMPORTANCE Rapid mutation rates and correspondingly high levels of intra- and interhost diversity are often cited as key features of viruses with the capacity for emergence and sustained transmission in a new host species. However, most of this information comes from studies of RNA viruses, with relatively little known about evolutionary processes in viruses with single-stranded DNA (ssDNA) genomes. Here, we provide a unique model of virus evolution, integrating both long-term global-scale and short-term intrahost evolutionary processes of an ssDNA virus that emerged to cause a pandemic in a new host animal. Our analysis reveals that successful host jumping and sustained transmission does not necessarily depend on a high level of intrahost diversity nor result in the continued accumulation of high levels of long-term evolution change. These findings indicate that all aspects of the biology and ecology of a virus are relevant when considering their adaptability.

Development of an avian avulavirus 1 (AAvV-1) L-gene real-time RT-PCR assay using minor groove binding probes for application as a routine diagnostic tool

Newcastle disease is a devastating disease of poultry caused by Newcastle disease virus (NDV), a virulent form of avian avulavirus 1 (AAvV-1). A rapid, sensitive and specific means for the detection of NDV is fundamental for the control of this notifiable transboundary virus. Although several real-time RT-PCR assays exist for the detection of AAvV-1, diagnostic sensitivity and specificities can be sub-optimal. In this study, we describe a modification to an existing AAvV-1 l-gene RT-PCR screening assay, where the original probe set was replaced with minor groove binding (MGB) probes, to create the MGB l-gene assay. The diagnostic sensitivity and specificity of this assay was evaluated against a broad panel of both Class I and Class II AAvV-1 viruses of diverse and representative lineages/genotypes in both clinical samples and amplified viruses, and compared with a number of previously published real-time RT-PCR screening assays for AAvV-1. The MGB l-gene assay outperformed all other assays in this assessment, with enhanced sensitivity and specificity, detecting isolates from a broad range of virus lineages/genotypes (including contemporaneously-circulating strains). The assay has also proved its value for screening original clinical samples for the presence of AAvV-1, thus providing an improved screening assay for routine detection of this notifiable disease agent.

A Novel Method to Identify Epigenetic Subclones with Increased Fitness from Genomic DNA Methylation Data of Lymphoma and Leukemia Patients

DNA methylation has been functionally implicated in X-inactivation, genomic imprinting, and silencing of transposable elements. DNA methylation also has a complex regulatory relationship with gene expression. Canonically, methylation around the promoters of tumor-suppressor genes induces gene-silencing, thereby representing a hit in the two-hit hypothesis for the development of cancer. Unfortunately, profiling studies conducted to determine how aberrant methylation may contribute to cancer progression are confounded by heterogeneity in the original clinical sample. Thus, though studies in patients with Acute Myeloid Leukemia (AML), Diffuse Large B-cell Lymphoma (DLBCL), and Chronic Lymphocytic Leukemia (CLL) have found that variation in detected methylation values from patients at diagnosis correlates with prognosis following therapy, they do not address which subclonal methylation events contribute to cancer progression.To address this concern, we developed a novel computational method to deconvolve the bisulfite sequencing data from a sample into its major methylation profiles and their respective prevalence in the sample. Our method, based on a modified Hidden Markov Model, effectively models the autocorrelations found in methylation data and outperforms existing algorithms. Our method was validated across a wide range of mixture simulations, where bisulfite sequencing reads from various different cell types were subsampled to form test samples that could be deconvolved. We were able to accurately (98%) distinguish distinct methylation patterns corresponding to the expected underlying subpopulations, such as for CD14 and CD22 in mixtures of germinal center B-cells and monocytes and for CD4 and CD8A in mixtures of CD4+ T-lymphocytes and CD8+ T-lymphocytes. These patterns also recapitulated differentially methylated regions (DMRs) identified by an independent DMR-caller.Given that our method does not rely on cell-type specific parameters and is therefore robust to all samples, to further validate and demonstrate the applicability of our method, we conducted Agilent Methyl-Seq on 5 primary DLBCL samples procured by the Lymphoma Core at the Siteman Cancer Center. As a positive control, our method identified differential methylation profiles at loci expected to differ from underlying CD19+ and CD4+ cells, which comprise a large majority of each sample. Our method also identified distinct methylation profiles not found in reference profiles from normal cell-types, suggesting these methylation profiles may be specific to DLBCL. To further validate these findings, we used single-cell bisulfite-sequencing at ten loci to demonstrate that the methylation profiles predicted by our method from the original sample are found in individual cells. We found several methylation patterns that only existed in a subset of CD19+ cells, which may represent distinct epigenetic subclones of DLBCL.Using our novel computational method, we next profiled the subclonal epigenetic architecture of publicly available (dbGaP) paired samples from patients with AML (n=137) at diagnosis and following therapy. We were able to not only identify subclonal methylation profiles that were specific to cancer but also find profiles at higher prevalence in patients at relapse compared to diagnosis. These methylation profiles, which were enriched for genes in cancer pathways as seen by Gene Set Enrichment Analysis, may confer fitness advantages for a cancer subclone to expand. We are currently conducting additional analyses to characterize the epigenetic regulatory circuits that contribute to our observed increase in subclonal fitness. In summary, we have developed a robust method to identify subclonal methylation changes that may contribute to cancer progression and prognosis, as seen in AML, and may lead to new avenues for improving treatment for patients with leukemia or lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-388: New clinical diagnostic approach for miRNA quantification using the Chlorella virus DNA ligase

Abstract miRNAs have an immense potential to serve as diagnostic, prognostic and prediction biomarkers in the whole field of oncology. Their utilization in liquid biopsy as a non-invasive diagnostic tool is very promising as well. However, to our knowledge, no miRNA-based diagnostics is currently used in clinical laboratories. We believe the reason is simple - the lack of easy, fast, sensitive and reproducible measurement technology, which would have passed the strict clinical validation criteria. On this count we have optimized a very promising miRNA detection technique, utilizing the enzyme Chlorella virus DNA ligase (SplintR® ligase, New England Biolabs), for clinical use. This two-step method involves ligation of two adjacent DNA oligonucleotides hybridized to a miRNA target, followed by TaqMan probe real-time quantitative PCR (qPCR). We managed to reduce the total procedure time from previous 5 hours to 2 hours and 15 minutes maintaining the high sensitivity (1 amol/µl in original clinical sample) and large dynamic range (7 logs). Moreover, we enabled the use of two PCR detection chemistries - more cost-effective SYBR green or more specific TaqMan probe. Based on this optimized method we developed an assay for detection of miR-142-5p, potential marker of colorectal cancer. We measured miR-142-5p profiles in the real samples of whole blood, plasma and serum of healthy donors. The results showed an excellent correlation with the TaqMan qPCR assay and miREIA® immuno-based technology for absolute quantification of miRNA. We conclude, that using SplintR® ligase for miRNA detection is potentially useful for clinical diagnostics. This work was funded by the Ministry of Industry and Trade of Czech Republic, project No. CZ.01.1.02/0.0/0.0/16_084/0008832. Citation Format: Iveta Hynstova, Tereza Mrackova, Michaela Adamcova, Barbora Dvorakova, Zlata Stastna, Ondrej Slaby, Milan Bartos, Viktor Ruzicka. New clinical diagnostic approach for miRNA quantification using the Chlorella virus DNA ligase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-388.

Ultrasensitive Capture of Human Herpes Simplex Virus Genomes Directly from Clinical Samples Reveals Extraordinarily Limited Evolution in Cell Culture.

Herpes simplex viruses (HSVs) are difficult to sequence due to their large DNA genome, high GC content, and the presence of repeats. To date, most HSV genomes have been recovered from culture isolates, raising concern that these genomes may not accurately represent circulating clinical strains. We report the development and validation of a DNA oligonucleotide hybridization panel to recover nearly complete HSV genomes at abundances up to 50,000-fold lower than previously reported. Using copy number information on herpesvirus and host DNA background via quantitative PCR, we developed a protocol for pooling for cost-effective recovery of more than 50 HSV-1 or HSV-2 genomes per MiSeq run. We demonstrate the ability to recover >99% of the HSV genome at >100× coverage in 72h at viral loads that allow whole-genome recovery from latently infected ganglia. We also report a new computational pipeline for rapid HSV genome assembly and annotation. Using the above tools and a series of 17 HSV-1-positive clinical swabs sent to our laboratory for viral isolation, we show limited evolution of HSV-1 during viral isolation in human fibroblast cells compared to the original clinical samples. Our data indicate that previous studies using low-passage-number clinical isolates of herpes simplex viruses are reflective of the viral sequences present in the lesion and thus can be used in phylogenetic analyses. We also detect superinfection within a single sample with unrelated HSV-1 strains recovered from separate oral lesions in an immunosuppressed patient during a 2.5-week period, illustrating the power of direct-from-specimen sequencing of HSV.IMPORTANCE Herpes simplex viruses affect more than 4 billion people across the globe, constituting a large burden of disease. Understanding the global diversity of herpes simplex viruses is important for diagnostics and therapeutics as well as cure research and tracking transmission among humans. To date, most HSV genomics has been performed on culture isolates and DNA swabs with high quantities of virus. We describe the development of wet-lab and computational tools that enable the accurate sequencing of near-complete genomes of HSV-1 and HSV-2 directly from clinical specimens at abundances >50,000-fold lower than previously sequenced and at significantly reduced cost. We use these tools to profile circulating HSV-1 strains in the community and illustrate limited changes to the viral genome during the viral isolation process. These techniques enable cost-effective, rapid sequencing of HSV-1 and HSV-2 genomes that will help enable improved detection, surveillance, and control of this human pathogen.

Ex-vivo assessment of drug response on breast cancer primary tissue with preserved microenvironments

ABSTRACTInteraction between cancerous, non-transformed cells, and non-cellular components within the tumor microenvironment plays a key role in response to treatment. However, short-term culture or xenotransplantation of cancer specimens in immunodeficient animals results in dramatic modifications of the tumor microenvironment, thus preventing reliable assessment of compounds or biologicals of potential therapeutic relevance.We used a perfusion-based bioreactor developed for tissue engineering purposes to successfully maintain the tumor microenvironment of freshly excised breast cancer tissue obtained from 27 breast cancer patients and used this platform to test the therapeutic effect of antiestrogens as well as checkpoint-inhibitors on the cancer cells.Viability and functions of tumor and immune cells could be maintained for over 2 weeks in perfused bioreactors. Next generation sequencing authenticated cultured tissue specimens as closely matching the original clinical samples. Anti-estrogen treatment of cultured estrogen receptor positive breast cancer tissue as well as administration of pertuzumab to a Her2 positive breast cancer both had an anti-proliferative effect. Treatment with anti-programmed-death-Ligand (PD-L)-1 and anti-cytotoxic T lymphocyte-associated protein (CTLA)-4 antibodies lead to immune activation, evidenced by increased lymphocyte proliferation, increased expression of IFNγ, and decreased expression of IL10, accompanied by a massive cancer cell death in ex vivo triple negative breast cancer specimens.In the era of personalized medicine, the ex vivo culture of breast cancer tissue represents a promising approach for the pre-clinical evaluation of conventional and immune-mediated treatments and provides a platform for testing of innovative treatments.

Using NSG recipient mice improves engraftment of gastric cancer patient derived xenografts.

70 Background: Previous gastric cancer (GC) patient derived xenograft (PDX) studies reported engraftment rates inversely proportional to the immunocompromised states of recipient mice (nudes 17%, scid 27%, and NOD/ scid 34%). We tested highly immunocompromised NOD scidgamma (NSG) mice, which lack mature T cells, B cells, or functional natural killer cells, as recipients for low volume biopsies and post-treatment GC samples. Methods: Consent was obtained from GC patients who were scheduled to undergo esophagogastroduodenoscopy (EGD) and diagnostic laparoscopy as part of their disease management strategies. The following amounts of tumor were coated with Matrigel and inoculated into the flanks of NSG mice: EGD biopsies ~10 mg, post-treatment samples ~100 mm3, and peritoneal metastasis biopsies ~100 mm3. Tumors were serially measured and passaged when the greatest dimension reached 1.5 cm, or if there was overlying skin necrosis. Results: The engraftment rates were: EGD biopsies 48% (13 of 27 samples), post-resection samples 58% (7 of 12), and peritoneal metastases 11% (1 of 9). Median time to first passage was 10.1 weeks (range: 8.1 to 12.4) for EGD biopsies and 22.1 weeks (9.6 to 33.0) for post-treatment samples. Two (15%) engrafted EGD mice developed liver metastases, and one (7.7%) had axillary lymph node metastasis (AxLN). Three (47%) engrafted post-treatment mice had liver metastases and two (29%) had AxLN. Histology was generally maintained through passages and metastases with some loss of mucinous components. There were no associations between engraftment rate and any evaluated clinical or pathologic characteristics, including tumor response and overall survival. The tumor from the one patient who had a complete pathologic response after NAT did not engraft. Conclusions: Using highly immunocompromised NSG mice improved engraftment rates of GC PDX, even for challenging specimens such as endoscopic biopsies and post-treatment resection samples. Peritoneal biopsies did not engraft well. Tumor histology was generally maintained through passages. Studies comparing the expression profiles of serially passaged tumors to the original clinical samples are ongoing.

Distinguishing highly similar gene isoforms with a clustering-based bioinformatics analysis of PacBio single-molecule long reads.

BackgroundGene isoforms are commonly found in both prokaryotes and eukaryotes. Since each isoform may perform a specific function in response to changing environmental conditions, studying the dynamics of gene isoforms is important in understanding biological processes and disease conditions. However, genome-wide identification of gene isoforms is technically challenging due to the high degree of sequence identity among isoforms. Traditional targeted sequencing approach, involving Sanger sequencing of plasmid-cloned PCR products, has low throughput and is very tedious and time-consuming. Next-generation sequencing technologies such as Illumina and 454 achieve high throughput but their short read lengths are a critical barrier to accurate assembly of highly similar gene isoforms, and may result in ambiguities and false joining during sequence assembly. More recently, the third generation sequencer represented by the PacBio platform offers sufficient throughput and long reads covering the full length of typical genes, thus providing a potential to reliably profile gene isoforms. However, the PacBio long reads are error-prone and cannot be effectively analyzed by traditional assembly programs.ResultsWe present a clustering-based analysis pipeline integrated with PacBio sequencing data for profiling highly similar gene isoforms. This approach was first evaluated in comparison to de novo assembly of 454 reads using a benchmark admixture containing 10 known, cloned msg genes encoding the major surface glycoprotein of Pneumocystis jirovecii. All 10 msg isoforms were successfully reconstructed with the expected length (~1.5 kb) and correct sequence by the new approach, while 454 reads could not be correctly assembled using various assembly programs. When using an additional benchmark admixture containing 22 known P. jirovecii msg isoforms, this approach accurately reconstructed all but 4 these isoforms in their full-length (~3 kb); these 4 isoforms were present in low concentrations in the admixture. Finally, when applied to the original clinical sample from which the 22 known msg isoforms were cloned, this approach successfully identified not only all known isoforms accurately (~3 kb each) but also 48 novel isoforms.ConclusionsPacBio sequencing integrated with the clustering-based analysis pipeline achieves high-throughput and high-resolution discrimination of highly similar sequences, and can serve as a new approach for genome-wide characterization of gene isoforms and other highly repetitive sequences.Electronic supplementary materialThe online version of this article (doi:10.1186/s13040-016-0090-8) contains supplementary material, which is available to authorized users.

Human cytomegalovirus: taking the strain.

In celebrating the 60th anniversary of the first isolation of human cytomegalovirus (HCMV), we reflect on the merits and limitations of the viral strains currently being used to develop urgently needed treatments. HCMV research has been dependent for decades on the high-passage strains AD169 and Towne, heavily exploiting their capacity to replicate efficiently in fibroblasts. However, the genetic integrity of these strains is so severely compromised that great caution needs to be exercised when considering their past and future use. It is now evident that wild-type HCMV strains are not readily propagated in vitro. HCMV mutants are rapidly selected during isolation in fibroblasts, reproducibly affecting gene RL13, the UL128 locus (which includes genes UL128, UL130 and UL131A) and often the UL/b′ region. As a result, the virus becomes less cell associated, altered in tropism and less pathogenic. This problem is not restricted to high-passage strains, as even low-passage strains can harbour biologically significant mutations. Cloning and manipulation of the HCMV genome as a bacterial artificial chromosome (BAC) offers a means of working with stable, genetically defined strains. To this end, the low-passage strain Merlin genome was cloned as a BAC and sequentially repaired to match the viral sequence in the original clinical sample from which Merlin was derived. Restoration of UL128L to wild type was detrimental to growth in fibroblasts, whereas restoration of RL13 impaired growth in all cell types tested. Stable propagation of phenotypically wild-type virus could be achieved only by placing both regions under conditional expression. In addition to the development of these tools, the Merlin transcriptome and proteome have been characterized in unparalleled detail. Although Merlin may be representative of the clinical agent, high-throughput whole-genome deep sequencing studies have highlighted the remarkable high level of interstrain variation present in circulating virus. There is a need to develop systems capable of addressing the significance of this diversity, free from the confounding effects of genetic changes associated with in vitro adaptation. The generation of a set of BAC clones, each containing the genome of a different HCMV strain repaired to match the sequence in the clinical sample, would provide a pathway to address the biological and clinical effects of natural variation in wild-type HCMV.

Molecular and serological findings in suspected patients with Crimean-Congo hemorrhagic fever virus in Iran.

Crimean-Congo hemorrhagic fever (CCHF) is an arthropod-borne disease of humans associated with a severe clinical picture, including hemorrhagic syndrome and a high mortality rate. CCHF virus is widely distributed throughout large areas of the world. To characterize the serological status in CCHF patients, paired clinical samples were collected from suspected CCHF patients and analyzed by microbiological and other laboratory analyses with the aim of: determining the presence of neutralizing antibodies against CCHF virus; investigating the cross-reactivity of these neutralizing antibodies against virus isolated from the same outbreak and against other available laboratory strain; and studying the relationship between the isolated virus with other virus by whole genome sequencing. Patients at Boo-Ali Hospital, Zahedan, Iran, with clinical symptoms ranging from mild to severe hemorrhagic fever were included in the study. Two serum samples were taken from each patient, the first as soon as the patient matched the criteria for CCHF notification and the second when the patient was discharged from hospital (2 weeks later). Commercial and in-house assays revealed a positive IgM signal in acute serum samples from six patients. A novel finding was that CCHF patients develop neutralizing antibodies soon after infection. Interestingly these antibodies were able to neutralize other CCHF virus strains too. The complete sequence of the Zahedan 2007 isolate, including the hitherto unknown first L-segment sequence, was identified using an original clinical sample from one patient with confirmed CCHF infection.

Psychometrics of the Symptoms and Functioning Severity Scale for High-Risk Youth.

Youth in residential care have significant mental health needs which require regular progress monitoring; however, very few emotional or behavioral assessments have been examined with this unique, high-risk population. This study examined the psychometrics of the Symptom Functioning and Severity Scale, a brief 24-item measure designed to assess the emotional and behavioral status of youth. This study examined the SFSS ratings from 143 youth with a disruptive behavior diagnosis living in a group-home facility in the Midwest and 52 of their service providers. Overall, the findings suggest that the psychometrics of the SFSS, when rated by staff or youth were similar to the original outpatient clinical samples. More specifically, the Rasch analyses indicate that the SFSS items and the overall scale is performing adequately, and the confirmatory factor analyses replicated the two-factor structure for staff. However, the fit of the two-factor model was less compelling for youth ratings. In all, the brief SFSS seems a promising measure for assessing problem severity for youth in residential care.

Antigenic drift of the pandemic 2009 A(H1N1) influenza virus in A ferret model.

Surveillance data indicate that most circulating A(H1N1)pdm09 influenza viruses have remained antigenically similar since they emerged in humans in 2009. However, antigenic drift is likely to occur in the future in response to increasing population immunity induced by infection or vaccination. In this study, sequential passaging of A(H1N1)pdm09 virus by contact transmission through two independent series of suboptimally vaccinated ferrets resulted in selection of variant viruses with an amino acid substitution (N156K, H1 numbering without signal peptide; N159K, H3 numbering without signal peptide; N173K, H1 numbering from first methionine) in a known antigenic site of the viral HA. The N156K HA variant replicated and transmitted efficiently between naïve ferrets and outgrew wildtype virus in vivo in ferrets in the presence and absence of immune pressure. In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties. The N156K escape mutant was antigenically distinct from wildtype virus as shown by binding of HA-specific antibodies. Glycan binding assays demonstrated the N156K escape mutant had altered receptor binding preferences compared to wildtype virus, which was supported by computational modeling predictions. The N156K substitution, and culture adaptations, have been detected in human A(H1N1)pdm09 viruses with N156K preferentially reported in sequences from original clinical samples rather than cultured isolates. This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection. Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance.