Clonal Evolution of Glioblastoma under Therapy methods
Aim. Evidence-backed execution summary for Clonal Evolution of Glioblastoma under Therapy methods from Clonal Evolution of Glioblastoma under Therapy.
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This experiment, in seven questions
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What do I need before I start?
human
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Cell culture, lentivirus production and cell growth analysis
reagent used in the protocol.
- Use
- After infection cells were selected with Puromycin (Sigma) at concentration of 2 mg/ml for 48 h. Cells were analysed by western blot, qRT-PCR and growth assay 3 days later.
Sequencing and mapping
reagent used in the protocol.
- Use
- Genomic DNA from initial tumor/recurrent tumor/matched normal blood of patients R001-R016, and recurrent tumor of patients R017-R019 were extracted purified, quantitated, fragmented, quality controlled and used to create a library of genomic DNA fragments. gDNA fragmentation was performed using the Covaris S220 AFA...
Cell culture, lentivirus production and cell growth analysis
reagent used in the protocol.
- Use
- Lentivirus was generated by co-transfection of the lentiviral vectors with pCMV-ΔCMV-ors wpMD2.G plasmids into HEK293T cells as previously described (Niola et al. JCI 2013; Carro et al. Nature 2010). shRNA sequences for LTBP4 are in.
Validation of mutations
reagent used in the protocol.
- Use
- The PCR products were purified with ExoSAP-IT (Affymetrix, USA) and subjected to Sanger Sequencing (Macrogen, USA). The amplicons containing the predicted genomic mutations were sequenced using BigDye Terminator Cycle Sequencing Kit v3.1 on the ABI Prism 3730 × l DNA Analyzer (Applied Biosystems, USA). All Sanger...
Cell culture, lentivirus production and cell growth analysis
reagent used in the protocol.
- Use
- U87 (ATCC HTB-14) cell line was acquired through American Type Culture Collection. U251 (Sigma, catalogue number 09063001) cell line was obtained through Sigma. Cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Sigma). Cells were routinely tested for mycoplasma contamination using Mycop...
Cell culture, lentivirus production and cell growth analysis
reagent used in the protocol.
- Use
- Evaluation of cell growth was performed using the MTT assay. Cells were plated at density of 2.5 × 10 3 cells/well into 96 well plates in 6 replicates and allowed to adhere for 24 h. Viability was assessed daily by adding MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium, Sigma 5mg/ml in PBS). Following...
RT-PCR
reagent used in the protocol.
- Use
- Total RNA was prepared with Trizol reagent (Invitrogen) and cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen) as described (Carro et al. Nature 2010; Zhao et al. Nature Cell Biol 2008). The quantitative RT-PCR was performed with 7500 Real-Time PCR system, using SYBR Green PCR Master...
Western Blot
reagent used in the protocol.
- Use
- Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium dexoycholate, 0.1% sodium dodecyl sulphate, 1.5 mM Na 3 VO 4, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β-glycerolphosphate and EDTA-free protease inhibitor cocktail (Roche)). Lysates were clear...
Clonal Replacement Events are Frequent in GBM
To discover the pattern of alterations in recurrent GBM compared with untreated tumors, we performed in-depth investigations into any gains or losses of genetic alterations. The epidermal growth factor receptor ( EGFR ) gene is known to be frequently amplified, mutated, and rearranged in untreated gliomas. To uncov...
- Use
- To discover the pattern of alterations in recurrent GBM compared with untreated tumors, we performed in-depth investigations into any gains or losses of genetic alterations. The epidermal growth factor receptor ( EGFR ) gene is known to be frequently amplified, mutated, and rearranged in untreated gliomas. To uncov...
Gene Fusion Detection and Structure Rearrangement of EGFR
To check the rearrangement of EGFR, we applied prada-guess-if from PRADA package. PRADA is a RNA sequencing analysis pipeline developed in MD Anderson. Following the definition in Brennan et al. 2013, transcribed allelic fractions of EGFRvIII were defined as the fraction of junction reads between exon1 and exon8.
- Use
- To check the rearrangement of EGFR, we applied prada-guess-if from PRADA package. PRADA is a RNA sequencing analysis pipeline developed in MD Anderson. Following the definition in Brennan et al. 2013, transcribed allelic fractions of EGFRvIII were defined as the fraction of junction reads between exon1 and exon8.
Sequencing and mapping
Genomic DNA from initial tumor/recurrent tumor/matched normal blood of patients R001-R016, and recurrent tumor of patients R017-R019 were extracted purified, quantitated, fragmented, quality controlled and used to create a library of genomic DNA fragments. gDNA fragmentation was performed using the Covaris S220 AFA...
- Use
- Genomic DNA from initial tumor/recurrent tumor/matched normal blood of patients R001-R016, and recurrent tumor of patients R017-R019 were extracted purified, quantitated, fragmented, quality controlled and used to create a library of genomic DNA fragments. gDNA fragmentation was performed using the Covaris S220 AFA...
Sequencing and mapping
Mapping files of TCGA samples but R039 were downloaded through CG-hub from TCGA. DNA mapping files of cohort UCSF, cohort MD Anderson, cohort KU, and R056-R093 from cohort SMC were all downloaded from European Genome-phenome Archive. Additional samples (R094-R114) from SMC followed the same sequencing protocols as t...
- Use
- Mapping files of TCGA samples but R039 were downloaded through CG-hub from TCGA. DNA mapping files of cohort UCSF, cohort MD Anderson, cohort KU, and R056-R093 from cohort SMC were all downloaded from European Genome-phenome Archive. Additional samples (R094-R114) from SMC followed the same sequencing protocols as t...
SAVI2 and driver gene selection
To identify somatic mutations from whole-exome sequencing data of triple samples (normal, initial tumor, and recurrent tumor) of GBM patients, we applied variance-calling software SAVI2 (statistical algorithm for variant frequency identification ) based on the empirical Bayesian method. Specifically, we first genera...
- Use
- To identify somatic mutations from whole-exome sequencing data of triple samples (normal, initial tumor, and recurrent tumor) of GBM patients, we applied variance-calling software SAVI2 (statistical algorithm for variant frequency identification ) based on the empirical Bayesian method. Specifically, we first genera...
SAVI2 and driver gene selection
Common somatic mutations from Patients R078-R082 were unknown due to the lack of normal DNA. The initial and recurrence exclusive mutations were calculated based on the difference between initial and recurrent tumor DNA. Tumor DNA of Patient R083-R093, R102, R111-R114 were not complete. The somatic mutations of thes...
- Use
- Common somatic mutations from Patients R078-R082 were unknown due to the lack of normal DNA. The initial and recurrence exclusive mutations were calculated based on the difference between initial and recurrent tumor DNA. Tumor DNA of Patient R083-R093, R102, R111-R114 were not complete. The somatic mutations of thes...
The Analysis of Loss of heterozygosity (LOH) and Copy Number Change
The pipeline of EXCAVATOR was carried out to detect copy number alterations based on whole-exome sequencing data. EXCAVATOR considers mean number of reads per exon, and normalized the data by a three-step normalization procedure to eliminate the bias introduced by GC content, the genomic mappability and the exon siz...
- Use
- The pipeline of EXCAVATOR was carried out to detect copy number alterations based on whole-exome sequencing data. EXCAVATOR considers mean number of reads per exon, and normalized the data by a three-step normalization procedure to eliminate the bias introduced by GC content, the genomic mappability and the exon siz...
Gene Fusion Detection and Structure Rearrangement of EGFR
ChimeraScan was used to generate the starting set of gene fusion candidates. To reduce the false positive rate and nominate potential driving events, we applied the Pegasus annotation and prediction pipeline. We reconstructed the entire fusion sequence on the basis of the breakpoint coordinates and assigned a driver...
- Use
- ChimeraScan was used to generate the starting set of gene fusion candidates. To reduce the false positive rate and nominate potential driving events, we applied the Pegasus annotation and prediction pipeline. We reconstructed the entire fusion sequence on the basis of the breakpoint coordinates and assigned a driver...
SAVI2 and driver gene selection
Software used for acquisition, scoring, statistics, or reporting.
- Use
- To identify somatic mutations from whole-exome sequencing data of triple samples (normal, initial tumor, and recurrent tumor) of GBM patients, we applied variance-calling software SAVI2 (statistical algorithm for variant frequency identification ) based on the empirical Bayesian method. Specifically, we first genera...
Before you run
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First confirmation
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Open the source paper before finalizing run-specific details.
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Open quote workflowStep-by-step procedure
What do I do, in order?
Cell culture, lentivirus production and cell growth analysis
After infection cells were selected with Puromycin (Sigma) at concentration of 2 mg/ml for 48 h. Cells were analysed by western blot, qRT-PCR and growth assay 3 days later.
METHODS
The specimens in cohort INCB originate from the Besta Brain Tumor Biobank, which is partly funded by the Italian Minister of Health. All patients signed an informed consent for the use of their biological material for research purposes. One case (R012) from this cohort had a history of lower grade glioma prior to the first GBM. All patients were treated by standard Stupp treatment with surgery followed by radiotherapy plus concomitant and adjuvant TMZ.
Reconstruction of the main routes of GBM evolution
Estimates of divergence time suggest that the recurrent clone diverged from the untreated clone many years before disease was detected ( ). The median among the 45 well-fitting patients had a divergence time of 12.6 years (range 2.3-50.5, IQR 7.2-22.6). Since the remaining 47 non-fitting patients may fail the model's assumption that untreated and recurrent tumors be evolutionarily distinct (i.e., monophyletic), we caution that divergence time for these patients be interpreted only as a heuristic measure of genetic difference between the two tumor samples. In general, uncertainty in the divergence time was large, with the median well-fitting patient showing a 95% CI 24 years wide. Still, even the bottom of the 95% CI exceeded three years for a majority of patients.
Cell culture, lentivirus production and cell growth analysis
Evaluation of cell growth was performed using the MTT assay. Cells were plated at density of 2.5 × 10 3 cells/well into 96 well plates in 6 replicates and allowed to adhere for 24 h. Viability was assessed daily by adding MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium, Sigma 5mg/ml in PBS). Following 4h incubation period, medium was removed and formazan crystal were solubilized with acidic isopropanol (0.1 N HCl in absolute isopropanol. The absorbance at 550 nm was measured with a plate reader.
Western Blot
Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium dexoycholate, 0.1% sodium dodecyl sulphate, 1.5 mM Na 3 VO 4, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β-glycerolphosphate and EDTA-free protease inhibitor cocktail (Roche)). Lysates were cleared by centrifugation at 15,000 r.p.m. for 15 min at 4 °C. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked in TBS with 5% non-fat milk and 0.1% Tween20, and probed with primary antibodies. Antibodies and working concentrations are: LTBP4 (1:200, sc-393666) obtained from Santa-Cruz Biotechnology; P-SMAD7 (1:1000, #3101) and SMAD7 (1:1000, #5339) obtained from Cell Signaling Technology; β-actin (1:2,000 dilution; A5441) obtained from Sigma.
Gene Fusion Validation
For validation of fusion transcripts and RT-PCR assays were performed. Total RNA was extracted from the tissues by AllPrep DNA/RNA Mini kit according to the manufacturer's instructions (Qiagen). The total RNA (0.5 µg) was reverse transcribed to synthesize template cDNA by a random primer using the SuperScriptIII First-Strand System(Life Technologies), and 20 µl synthesized cDNA was diluted 10 times with DW. For RT-PCR, EzWay Taq PCR MasterMix (Komabiotech, KOREA) and 5 µl synthesized cDNA as template were used. Thermal cycling was carried out under the following conditions: 1 min at 95°C followed by 30 cycles of 30 sec at 95°C, 30 sec at 55°C, 30 sec at 72°C. The primer pairs used in this experiment were designed to make the amplification product including the breakpoints of the fusion genes. PCR products were analyzed by agarose gel electropho...
Measurement outputs
What raw and processed outputs should exist?
After infection cells were selected with Puromycin (Sigma) at concentration of 2 mg/ml for 48 h. Cells were analysed by western blot, qRT-PCR and growth assay 3 days later.
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
As indicated in, 17% of TMZ treated GBM patients (17/100) relapsed with hypermutated tumors, yet there is no incidence of hypermutation in non-TMZ treated patients (0/14). The...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
To reveal the potential evolutionary trajectories of GBM under therapy a tumor evolutionary directed graph (TEDG) was constructed for the 93 triplet samples. As this analysis us...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Based on its pattern of gene expression, GBM is commonly divided into four subtypes, which display different responses to treatment. To study evolution of gene expression in GB...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
As indicated in, 17% of TMZ treated GBM patients (17/100) relapsed with hypermutated tumors, yet there is no incidence of hypermutation in non-TMZ treated patients (0/14).
from paperScoring or quantification
Quantify the primary readouts for this experiment: After infection cells were selected with Puromycin (Sigma) at concentration of 2 mg/ml for 48 h. Cells were analysed by western blot, qRT-PCR and growth assay 3 days later.; As indicated in, 17% of TMZ treated GBM patients (17/100) relapsed with hypermutated tumors, yet there is no incidence of hypermutation in non-TMZ treated patients (0/14). The...; To reveal the potential evolutionary trajectories of GBM under therapy a tumor evolutionary directed graph (TEDG) was constructed for the 93 triplet samples. As this analysis us...; Based on its pattern of gene expression, GBM is commonly divided into four subtypes, which display different responses to treatment. To study evolution of gene expression in GB....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
As indicated in, 17% of TMZ treated GBM patients (17/100) relapsed with hypermutated tumors, yet there is no incidence of hypermutation in non-TMZ treated patients (0/14). The...; The number of mutations exclusive to untreated tumors, recurrent tumors, or those in common can be used to describe an evolutionary tree. We developed a method to perform statis...; By accounting for the likelihood of each mutational pattern within our branching model, we fit substitution rates before and after treatment, as well as the amount of time befor...; To identify somatic mutations from whole-exome sequencing data of triple samples (normal, initial tumor, and recurrent tumor) of GBM patients, we applied variance-calling softwa...
from paperReporting output
Report representative outputs alongside summary comparisons for After infection cells were selected with Puromycin (Sigma) at concentration of 2 mg/ml for 48 h. Cells were analysed by western blot, qRT-PCR and growth assay 3 days later., As indicated in, 17% of TMZ treated GBM patients (17/100) relapsed with hypermutated tumors, yet there is no incidence of hypermutation in non-TMZ treated patients (0/14). The..., To reveal the potential evolutionary trajectories of GBM under therapy a tumor evolutionary directed graph (TEDG) was constructed for the 93 triplet samples. As this analysis us..., Based on its pattern of gene expression, GBM is commonly divided into four subtypes, which display different responses to treatment. To study evolution of gene expression in GB....
inferred from protocolStructured statistical methods
As indicated in, 17% of TMZ treated GBM patients (17/100) relapsed with hypermutated tumors, yet there is no incidence of hypermutation in non-TMZ treated patients (0/14). The...; The number of mutations exclusive to untreated tumors, recurrent tumors, or those in common can be used to describe an evolutionary tree. We developed a method to perform statis...; By accounting for the likelihood of each mutational pattern within our branching model, we fit substitution rates before and after treatment, as well as the amount of time befor...; To identify somatic mutations from whole-exome sequencing data of triple samples (normal, initial tumor, and recurrent tumor) of GBM patients, we applied variance-calling softwa...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
After infection cells were selected with Puromycin (Sigma) at concentration of 2 mg/ml for 48 h. Cells were analysed by western blot, qRT-PCR and growth assay 3 days later.
The specimens in cohort INCB originate from the Besta Brain Tumor Biobank, which is partly funded by the Italian Minister of Health. All patients signed an informed consent for the use of their biological material for research purposes. One case (R012) from this cohort had a history of lower grade glioma prior to the first GBM. All patients were treated by standard Stupp treatment with surgery followed by radiotherapy plus concomitant and adjuvant TMZ.
Estimates of divergence time suggest that the recurrent clone diverged from the untreated clone many years before disease was detected ( ). The median among the 45 well-fitting patients had a divergence time of 12.6 years (range 2.3-50.5, IQR 7.2-22.6). Since the remaining 47 non-fitting patients may fail the model's assumption that untreated and recurrent tumors be evolutionarily distinct (i.e., monophyletic), we caution that divergence time for these patients be interpreted only as a heuristic measure of genetic difference between the two tumor samples. In general, uncertainty in the divergence time was large, with the median well-fitting patient showing a 95% CI 24 years wide. Still, even the bottom of the 95% CI exceeded three years for a majority of patients.
Evaluation of cell growth was performed using the MTT assay. Cells were plated at density of 2.5 × 10 3 cells/well into 96 well plates in 6 replicates and allowed to adhere for 24 h. Viability was assessed daily by adding MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium, Sigma 5mg/ml in PBS). Following 4h incubation period, medium was removed and formazan crystal were solubilized with acidic isopropanol (0.1 N HCl in absolute isopropanol. The absorbance at 550 nm was measured with a plate reader.
Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium dexoycholate, 0.1% sodium dodecyl sulphate, 1.5 mM Na 3 VO 4, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β-glycerolphosphate and EDTA-free protease inhibitor cocktail (Roche)). Lysates were cleared by centrifugation at 15,000 r.p.m. for 15 min at 4 °C. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked in TBS with 5% non-fat milk and 0.1% Tween20, and probed with primary antibodies. Antibodies and working concentrations are: LTBP4 (1:200, sc-393666) obtained from Santa-Cruz Biotechnology; P-SMAD7 (1:1000, #3101) and SMAD7 (1:1000, #5339) obtained from Cell Signaling Technology; β-actin (1:2,000 dilution; A5441) obtained from Sigma.
For validation of fusion transcripts and RT-PCR assays were performed. Total RNA was extracted from the tissues by AllPrep DNA/RNA Mini kit according to the manufacturer's instructions (Qiagen). The total RNA (0.5 µg) was reverse transcribed to synthesize template cDNA by a random primer using the SuperScriptIII First-Strand System(Life Technologies), and 20 µl synthesized cDNA was diluted 10 times with DW. For RT-PCR, EzWay Taq PCR MasterMix (Komabiotech, KOREA) and 5 µl synthesized cDNA as template were used. Thermal cycling was carried out under the following conditions: 1 min at 95°C followed by 30 cycles of 30 sec at 95°C, 30 sec at 55°C, 30 sec at 72°C. The primer pairs used in this experiment were designed to make the amplification product including the breakpoints of the fusion genes. PCR products were analyzed by agarose gel electrophoresis. The primers were summarized in.
Machine-readable layer
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"text": "Estimates of divergence time suggest that the recurrent clone diverged from the untreated clone many years before disease was detected ( ). The median among the 45 well-fitting patients had a divergence time of 12.6 years (range 2.3-50.5, IQR 7.2-22.6). Since the remaining 47 non-fitting patients may fail the model's assumption that untreated and recurrent tumors be evolutionarily distinct (i.e., monophyletic), we caution that divergence time for these patients be interpreted only as a heuristic measure of genetic difference between the two tumor samples. In general, uncertainty in the divergence time was large, with the median well-fitting patient showing a 95% CI 24 years wide. Still, even the bottom of the 95% CI exceeded three years for a majority of patients."
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