scholarly journals Patient Derived Xenograft (PDX) Models Recapitulate the Genomic-Driver Composition of Acute Leukemia Samples

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 286-286 ◽  
Author(s):  
Andrei V. Krivtsov ◽  
Xujun Wang ◽  
Noushin Rahnamay Farnoud ◽  
Michael Hadler ◽  
Marta Sanchez Martin ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Genomic Diversity ◽  
Driver Mutations ◽  
Rna Seq ◽  
Median Latency ◽  
Rna Molecules ◽  
Immunodeficient Mice ◽  
Pathogenic Variants ◽  
Nsg Mouse ◽  
Pdx Models

Abstract Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genomic lesions. However, given recent recognition that multiple genomically distinct sub-clones can exist in AML, there is a risk that there may be selection for sub-clones from the transplanted sample that might not fully represent the patient’s disease. We transplanted 160 (70 T-ALL 56 AML, 32 B-ALL, 2 MLL) patient samples of which 120 engrafted into at least 1 irradiated NSG mouse. 45 AML samples engrafted with a median latency 107+/-41 days. Transplantation of 6 PDX AML samples resulted in immunophenotypically identical disease within 87+/-35 days. 2 MLL samples engrafted in 100% of mice with a median latency of 103+/-13 days. 25 B-ALL samples engrafted with a median latency of 95+/-44 days. Secondary transplantation of 3 PDX B-ALL samples resulted in engraftment of leukemia cells with an identical immunophenotype in 100% of transplanted mice within 52+/-3 days. 48 T-ALL samples engrafted in at least one mouse within 50 days. Secondary transplantation of a single T-ALL PDX sample resulted in 100% engraftment within 31+/-10 days. Genomic DNA and total RNA were isolated from 150 (AML: 16Pt+33PDX; MLL 2Pt+6PDX; B-ALL 17Pt+38PDX; T-ALL 19Pt+19PDX) samples. Adaptor ligated sequencing libraries were captured by solution hybridization using baitsets for 405 cancer-related genes and selected introns for 31 genes frequently rearranged for DNA-seq, and 405 cancer-related and 265 genes frequently rearranged for RNA-seq. All libraries were sequenced averaging >500x for DNA and >6M total pairs for RNA (HiSeq). We detected on average 23+/-12 including a mean 5+/-4 known pathogenic variants such as CDKN2A/B deletion (20/13); FLT3 (SNV & -ITD) and NOTCH (11 ea); WT1 and TP53 (10 ea); NRAS (9); PTPN11 (7); NPM1c, PTEN, and KRAS (6) DNMT3A, IDH1/2, and ASXL1 (5 ea); FBXW7, CEBPA, and TET2 (4 ea); PHF6 and NF1 (3 ea); IKZF1, ATM, and JAK2 (2 ea). Analyses of fusion RNA molecules detected known fusions: MLL-AF4 (4); MLL-AF9 (2), CRLF2-P2RY8, ETV6-RUNX1 or TEL-AML1, PBX1-TCF3 (2 ea); MLL-AF10, MLL-ELL, MLL-EP300, MLL-PTD, BCR-ABL, BCL2-IGK, MYH11-CBFB, along with novel fusions: TCF3-OAZ1, RB1-RCBTB2, PAX5-FLI1, and PAX5-MSI2. The mutations found in the 54 patient samples were consistently identified in the 96 PDX, however some cases showing variation in allele frequency between diagnostic and engrafted samples. Collectively, all 1420 and 288 disease relevant variant allele frequency (VAF) correlated significantly between patient and PDX samples (R2=0.55, R2=0.43), respectively. We then assessed VAF changes from diagnostic to PDX sample as a measure of clonal concordance. Diagnostic and PDX sample were considered discordant if at least one disease relevant VAF demonstrated significant variation between these samples, accounted for small variability of infrequent variances considering SD of sequencing detection. 31 samples were scored as concordant and 23 as discordant which were similarly distributed between disease lineages and did not correlate with diseases status, future relapse or overall survival. Using the same rules we further accessed concordance only between PDX samples in 23 cases when patient samples were transplanted into multiple mice. All 10 groups of PDX samples that were concordant with patient samples were also concordant within the groups. 5 groups of PDX samples that were discordant with patient samples were concordant within groups. 8 groups of PDX samples that were discordant with patient samples were also discordant within their groups. Overall 15 samples produced concordant engraftment in mice and 8 samples produced discordant engraftment. We hypothesized that specific genomic lesions in the 8 groups might underline this discordance. Mutations of FLT3, RAS, TP53, PTPN11 and NOTCH1 correlated with clonal discordance. These findings show that the leukemias that are engrafted in mice mirror the genomic diversity of primary leukemia samples, and that the majority of PDX samples have a genotype similar to that observed in the clinical isolate. More importantly, our data demonstrate the feasibility of developing a large, genetically annotated bank of PDX leukemia models that can be used to test and credential novel therapeutics that target driver mutations in different leukemia subsets. Disclosures Stein: Seattle Genetics, Inc.: Research Funding; Janssen Pharmaceuticals: Consultancy. Wang:Foundation Medicine Inc: Employment. Miller:Foundation Medicine: Employment. Armstrong:Epizyme: Consultancy.

Patient Derived Xenograft (PDX) Models Faithfully Recapitulate The Genetic Composition Of Primary AML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1328-1328 ◽  
Author(s):  
Andrei V. Krivtsov ◽  
Wang Xujun ◽  
Noushin Rahnamay Farnoud ◽  
Michael Hadler ◽  
Kristina M. Knapp ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Limit Of Detection ◽  
Refractory Disease ◽  
Illumina Hiseq ◽  
Immunodeficient Mice ◽  
Patient Sample ◽  
First Case ◽  
Diagnostic Samples ◽  
Clonal Progression ◽  
Pdx Models

Abstract Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genetic lesions. However, given our recent recognition that multiple genetically distinct subclones can exist in AML, there is a risk that there may be selection for sub-clones from the xenotransplanted sample that might not fully represent the patient’s disease. We sought to establish a collection of genetically defined AML samples capable of engraftment in immunodeficient mice. We transplanted 30 AML patient samples; within 150 days (median 91 days) post transplantation 12 samples produced human CD45+ CD33+ CD19- CD3- engraftment in one or multiple NSG mice. Median patient sample amplification in 25 mice was 21 fold. Genomic DNA and total RNA was isolated from 7 AML patient samples (3 diagnostic samples from patients who remain in remission; 2 diagnostic samples from patients who later relapsed, 2 diagnostic samples from patients with refractory disease) and 14 matched xenotransplanted samples (2 mice per patient sample). Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >499x for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The mutations found in the 7 diagnostic samples were consistently identified in the 14 engrafted AML samples, but with some cases showing variation in allele frequency between diagnostic and engrafted samples. This finding shows that the human disease that engrafted in mice mimics the genetic makeup of the disease found in patients. We then assessed for allele frequency (AF) changes from diagnostic to xenografted sample as a measure of clonal progression. Clonal progression was defined as emergence of a clone carrying a novel genetic variant in the xenografted sample as compared to the diagnostic patient sample. Five patient samples (from 10 mice) did not show emergence of novel genetic lesions. In this group 2 patients had refractory disease and 3 patients remain in remission. Two patient samples (from 4 mice) demonstrated apparent emergence of novel genetic lesions not detected in diagnostic patient samples. Both of these patients have relapsed since the diagnostic samples were acquired. In the first case, both xenotransplanted mice engrafted with disease carrying NRAS N12S mutation (AF 0.05 and 0.09), which subsequent evaluation revealed to be present below the limit-of-detection (AF 0.004) in the clinical isolate obtained from patient presentation. We are currently conducting the same analysis on the relapsed sample from this patient. In the second case, both mice engrafted with disease carrying PTPN11 E76V (AF 0.03 and 0.0016) while the patient diagnostic sample did not contain any evidence of the alteration at 718x unique sequence coverage. Of note, one xenografted sample had an IDH1 R132C and another had IDH2 R140Q mutation, both of which have previously been shown to play a role in AML pathogenesis. Available AML cell lines do not carry IDH1/2 mutations, making it challenging to test IDH1/2 inhibitors in pre-clinical settings. These xenografted samples offer an opportunity to test such inhibitors. Overall we conclude that the xenotransplanted samples possess the diversity of genetic abnormalities found in diagnostic AML samples and thus can be used to assess efficacy of novel targeted therapies. We would like to further investigate a model in which the absence of clonal progression in xenografted samples would predict a better patient outcome, while emergence of novel clones might indicate an increased potential for relapse. We are currently expanding the study to include more diagnostic, xenotransplanted and relapsed samples to assess the associations between the ability of a sample to engraft in mice with clinical outcome and genetic/epigenetic lesions. Disclosures: Armstrong: Epizyme Inc.: Has consulted for Epizyme Inc. Other.

The FGFR-inhibitor derazantinib (DZB) is active in PDX-models of GI-cancer with specific aberrations in FGFR.

2020 ◽  
Vol 38 (4_suppl) ◽  
pp. 421-421
Author(s):  
Paul McSheehy ◽  
Felix Bachmann ◽  
Nicole Forster-Gross ◽  
Mahmoud El Shemerly ◽  
Mila Roceri ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Lines ◽  
Mapk Pathway ◽  
Growth Factor Receptor ◽  
Human Tumor ◽  
High Expression ◽  
Driver Mutations ◽  
Clinical Activity ◽  
Rna Seq ◽  
Pdx Models

421 Background: DZB is an oral small-molecule Fibroblast Growth Factor Receptor 1/2/3 inhibitor (FGFRi) with clinical activity in FGFR2-fusion-positive cholangiocarcinoma. DZB was screened for activity in gastrointestinal cancer (GIC), by using a panel of GIC cell-lines, human tumor xenografts and 30 GIC patient-derived xenograft (PDX) models. Methods: DZB anti-proliferative potency was determined in 26 GIC cell lines to determine the GI50. The GIC cell-line, SNU-16 was grown s.c. in nude mice and treated daily for 3-weeks with DZB at the MTD of 75 mg/kg, p.o. Plasma and tumor were removed and analyzed for drug-levels and PD biomarkers to assess pathway inhibition. DZB (@MTD) was tested in the PDX-screen (15 biliary, 13 gastric and 2 colorectal cancer; n≥3/group) using models with FGFR-fusions, FGFR-mutations and/or differing FGFR copy-number (CN)/RNA-seq expression levels. Efficacy and tolerability were quantified as a dT/C (treated/control). Results: Cellular GI50s ranged from 0.02-20 μM; the most sensitive (GI50≤0.5 μM) had FGFR-fusions or high-expression. In mice, DZB induced stasis of SNU-16 tumors (dT/C∼0.0) and was well tolerated (dT/C > 1.0); the plasma PK was dose-dependent with a Cmax of 2 μM (4 hr), a Cmin of 0.5 μM. DZB induced dose- and time-dependent changes in the MAPK-pathway and expression of downstream genes, consistent with its mode of action. In PDX-models, efficacy varied from no-response to 100% regression. Known driver-mutations were associated with partial-responses (best dT/C = 0.42), but models with FGFR-fusions, especially FGFR2-fusions, were very sensitive leading to stasis or strong-regression, particularly in gastric cancer. High-expression of FGFR2 was also associated with strong responses. There was no direct correlation between CN and high RNA-seq values suggesting amplification was not always a predictor of high expression. Endpoint PD-analyses of the PDX-models is ongoing to identify other potential stratifiers and PD-markers of response. Conclusions: DZB showed convincing activity in GIC-models with FGFR-fusions and/or high expression. A clinical trial is planned in patients with gastric cancer to investigate DZB as mono- and combination-therapy.

scholarly journals PSII-22 Comparison of the meat quality traits of crossbred pigs and the Large Black pigs

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 241-242
Author(s):  
Yongjie Wang ◽  
Keshari Thakali ◽  
Sarah Shelby ◽  
Jason Apple ◽  
Yan Huang
Keyword(s):  
Meat Quality ◽  
Genomic Diversity ◽  
Longissimus Dorsi ◽  
Rna Seq ◽  
Longissimus Dorsi Muscle ◽  
A Value ◽  
Dorsi Muscle ◽  
Body Weights ◽  
C Value ◽  
Crossbred Pigs

Abstract The purpose of this study was to compare the meat quality and genomic differences between cross-bred commercial pig (CP) and domestic Large Black pig (BP). Seven cross-bred commercial pigs and eight British Large Black pigs were assigned to CP group and BP group, with initial mean body weights of 18.82±1.412 kg for CP group and 23.31±1.935 kg for BP group, P = 0.061, and fed ad libitum. The final BW of the CP at d101 was similar to the BP (130.0±8.16 kg vs. 121.1±2.80 kg, P = 0.132). However, the BP group took 108 days to reach the final BW. The ADG in the CP was higher than BP (1.102±0.0599 kg vs. 0.905±0.0138 kg, P = 0.003). The hot carcass weight of CP was higher (P < 0.01) than BP, but the backfat of BP was higher (P < 0.01) than CP. The a* value of CP was higher (P < 0.05) than BP, and the c* value of CP was tended to be higher (P < 0.10) than BP. However, the h value of BP was higher (P < 0.05) than CP. The longissimus dorsi muscle fat content of BP was higher (P < 0.05) than CP. For the fatty acid composition, the SFA and MUFA of BP were higher (P < 0.05) than CP, but the PUFA of CP was higher (P < 0.05) than BP. The metmyoglobin content of CP was tended to be higher (P < 0.10) than BP. For the meat metabolism, the oxygen consumption of longissimus dorsi muscle of BP was higher (P < 0.01) than CP. The RNA-Seq data showed that the expression of the genes related to lipid metabolism is higher in BP (fold change > 3, P < 0.05). To conclude, BP has higher meat quality, while CP has its advantages in growth performance. And the differences between these two breeds may due to the genomic diversity.

scholarly journals nanotatoR: a tool for enhanced annotation of genomic structural variants

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Surajit Bhattacharya ◽  
Hayk Barseghyan ◽  
Emmanuèle C. Délot ◽  
Eric Vilain
Keyword(s):  
De Novo ◽  
Genome Mapping ◽  
Gene List ◽  
Sufficient Information ◽  
Rna Seq ◽  
Structural Variants ◽  
De Novo Genome Assembly ◽  
Pathogenic Variants ◽  
Increased Sensitivity

Abstract Background Whole genome sequencing is effective at identification of small variants, but because it is based on short reads, assessment of structural variants (SVs) is limited. The advent of Optical Genome Mapping (OGM), which utilizes long fluorescently labeled DNA molecules for de novo genome assembly and SV calling, has allowed for increased sensitivity and specificity in SV detection. However, compared to small variant annotation tools, OGM-based SV annotation software has seen little development, and currently available SV annotation tools do not provide sufficient information for determination of variant pathogenicity. Results We developed an R-based package, nanotatoR, which provides comprehensive annotation as a tool for SV classification. nanotatoR uses both external (DGV; DECIPHER; Bionano Genomics BNDB) and internal (user-defined) databases to estimate SV frequency. Human genome reference GRCh37/38-based BED files are used to annotate SVs with overlapping, upstream, and downstream genes. Overlap percentages and distances for nearest genes are calculated and can be used for filtration. A primary gene list is extracted from public databases based on the patient’s phenotype and used to filter genes overlapping SVs, providing the analyst with an easy way to prioritize variants. If available, expression of overlapping or nearby genes of interest is extracted (e.g. from an RNA-Seq dataset, allowing the user to assess the effects of SVs on the transcriptome). Most quality-control filtration parameters are customizable by the user. The output is given in an Excel file format, subdivided into multiple sheets based on SV type and inheritance pattern (INDELs, inversions, translocations, de novo, etc.). nanotatoR passed all quality and run time criteria of Bioconductor, where it was accepted in the April 2019 release. We evaluated nanotatoR’s annotation capabilities using publicly available reference datasets: the singleton sample NA12878, mapped with two types of enzyme labeling, and the NA24143 trio. nanotatoR was also able to accurately filter the known pathogenic variants in a cohort of patients with Duchenne Muscular Dystrophy for which we had previously demonstrated the diagnostic ability of OGM. Conclusions The extensive annotation enables users to rapidly identify potential pathogenic SVs, a critical step toward use of OGM in the clinical setting.

scholarly journals Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 964
Author(s):  
Sarka Benesova ◽  
Mikael Kubista ◽  
Lukas Valihrach
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
High Sensitivity ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Liquid Biopsies ◽  
Comprehensive Overview ◽  
Rna Molecules ◽  
Novel Mirna ◽  
The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

scholarly journals Allele frequency of variants reported to cause adenine phosphoribosyltransferase deficiency

2021 ◽  
Author(s):  
Hrafnhildur L. Runolfsdottir ◽  
John A. Sayer ◽  
Olafur S. Indridason ◽  
Vidar O. Edvardsson ◽  
Brynjar O. Jensson ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Autosomal Recessive Disorder ◽  
European Population ◽  
Adenine Phosphoribosyltransferase ◽  
Genomic Databases ◽  
Pathogenic Variants ◽  
Adenine Phosphoribosyltransferase Deficiency ◽  
Variation Database ◽  
The Uk ◽  
Aprt Deficiency

AbstractAdenine phosphoribosyltransferase deficiency is a rare, autosomal recessive disorder of purine metabolism that causes nephrolithiasis and progressive chronic kidney disease. The small number of reported cases indicates an extremely low prevalence, although it has been suggested that missed diagnoses may play a role. We assessed the prevalence of APRT deficiency based on the frequency of causally-related APRT sequence variants in a diverse set of large genomic databases. A thorough search was carried out for all APRT variants that have been confirmed as pathogenic under recessive mode of inheritance, and the frequency of the identified variants examined in six population genomic databases: the deCODE genetics database, the UK Biobank, the 100,000 Genomes Project, the Genome Aggregation Database, the Human Genetic Variation Database and the Korean Variant Archive. The estimated frequency of homozygous genotypes was calculated using the Hardy-Weinberg equation. Sixty-two pathogenic APRT variants were identified, including six novel variants. Most common were the missense variants c.407T>C (p.(Met136Thr)) in Japan and c.194A>T (p.(Asp65Val)) in Iceland, as well as the splice-site variant c.400 + 2dup (p.(Ala108Glufs*3)) in the European population. Twenty-nine variants were detected in at least one of the six genomic databases. The highest cumulative minor allele frequency (cMAF) of pathogenic variants outside of Japan and Iceland was observed in the Irish population (0.2%), though no APRT deficiency cases have been reported in Ireland. The large number of cases in Japan and Iceland is consistent with a founder effect in these populations. There is no evidence for widespread underdiagnosis based on the current analysis.

scholarly journals A comprehensive evaluation of pathogenic mutations in primary cutaneous melanomas, including the identification of novel loss-of-function variants

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ivana Ticha ◽  
Jan Hojny ◽  
Romana Michalkova ◽  
Ondrej Kodet ◽  
Eva Krkavcova ◽  
...  
Keyword(s):  
Cutaneous Melanoma ◽  
Comprehensive Evaluation ◽  
Mapk Pathway ◽  
Driver Mutations ◽  
Molecular Testing ◽  
Loss Of Function ◽  
Superficial Spreading ◽  
Pathogenic Variants ◽  
Frequent Mutations ◽  
Cycle Regulation

AbstractThe most common histological subtypes of cutaneous melanoma include superficial spreading and nodular melanoma. However, the spectrum of somatic mutations developed in those lesions and all potential druggable targets have not yet been fully elucidated. We present the results of a sequence capture NGS analysis of 114 primary nodular and superficial spreading melanomas identifying driver mutations using biostatistical, immunohistochemical and/or functional approach. The spectrum and frequency of pathogenic or likely pathogenic variants were identified across 54 evaluated genes, including 59 novel mutations, and the newly identified TP53 loss-of-function mutations p.(L194P) and p.(R280K). Frequently mutated genes most commonly affected the MAPK pathway, followed by chromatin remodeling, and cell cycle regulation. Frequent aberrations were also detected in the genes coding for proteins involved in DNA repair and the regulation and modification of cellular tight junctions. Furthermore, relatively frequent mutations were described in KDR and MET, which represent potential clinically important targets. Those results suggest that with the development of new therapeutic possibilities, not only BRAF testing, but complex molecular testing of cutaneous melanoma may become an integral part of the decision process concerning the treatment of patients with melanoma.

scholarly journals Factorial study of the RNA-seq computational workflow identifies biases as technical gene signatures

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Joël Simoneau ◽  
Ryan Gosselin ◽  
Michelle S Scott
Keyword(s):  
Factorization Method ◽  
Rna Seq ◽  
Similar Sequence ◽  
Gene Group ◽  
Rna Molecules ◽  
Design Choice ◽  
Rna Biology ◽  
Computational Workflow ◽  
The Common ◽  
Genome Annotations

Abstract RNA-seq is a modular experimental and computational approach aiming in identifying and quantifying RNA molecules. The modularity of the RNA-seq technology enables adaptation of the protocol to develop new ways to explore RNA biology, but this modularity also brings forth the importance of methodological thoroughness. Liberty of approach comes with the responsibility of choices, and such choices must be informed. Here, we present an approach that identifies gene group-specific quantification biases in current RNA-seq software and references by processing datasets using diverse RNA-seq computational pipelines, and by decomposing these expression datasets with an independent component analysis matrix factorization method. By exploring the RNA-seq pipeline using this systemic approach, we identify genome annotations as a design choice that affects to the same extent quantification results as does the choice of aligners and quantifiers. We also show that the different choices in RNA-seq methodology are not independent, identifying interactions between genome annotations and quantification software. Genes were mainly affected by differences in their sequence, by overlapping genes and genes with similar sequence. Our approach offers an explanation for the observed biases by identifying the common features used differently by the software and references, therefore providing leads for the betterment of RNA-seq methodology.

Sign in / Sign up

Export Citation Format

Share Document