Comprehensive genomic profiles of small cell lung cancer methods
Aim. Evidence-backed execution summary for Comprehensive genomic profiles of small cell lung cancer methods from Comprehensive genomic profiles of small cell lung cancer.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
DNA and RNA extractions
reagent used in the protocol.
- Use
- DNA was extracted from fresh-frozen tissues, EDTA blood, or FFPE samples using the Gentra Puregene DNA extraction kit (Qiagen) following the protocol of the manufacturer. DNA isolates were hydrated in TE-buffer and confirmed to be of high molecular weight (>10 kb) by agarose gel electrophoresis. Genomic DNA from fre...
DNA and RNA extractions
reagent used in the protocol.
- Use
- For RNA extractions, tissue sections were first lysed and homogenized with the Tissue Lyzer (Qiagen). Subsequent RNA extractions were performed with the Qiagen RNAeasy Mini Kit according to the instructions of the manufacturer. The RNA quality was assessed at the Bioanalyzer 2100 DNA Chip 7500 (Agilent Technologies)...
EdU incorporation assay
reagent used in the protocol.
- Use
- Transfected cells were treated with 10 µM EdU (5-ethynyl-2′-deoxyuridine) (Life Technologies) for 3 h before trypsinization for FACS. 1 × 10 5 live, GFP + cells were sorted and labelled with EdU using the Click-iT EdU Pacific Blue flow cytometry assay kit (Life Technologies). Cells were then run thro...
Recurrent somatic alterations in SCLC
reagent used in the protocol.
- Use
- Given the lack of therapeutic options in SCLC, we sought mutations that are known oncogenic drivers in other cancers and sometimes associated with response to targeted drugs (V),. Of these, we found mutations in four tumours with a potential therapeutic implication, including mutations in BRAF, KIT, and PIK3CA (...
Tumour suppressive roles of Notch in SCLC
reagent used in the protocol.
- Use
- In an unsupervised hierarchical clustering analysis of transcriptome sequencing data (Methods), we observed two major clusters of SCLC tumours ( and ). The majority (77%, n = 53/69) of tumours exhibited high expression of the neuroendocrine markers CHGA (chromogranin A) and GRP (gastrin releasing peptide), had high...
Next-generation sequencing
reagent used in the protocol.
- Use
- Whole-genome sequencing was performed with DNA extracted from fresh-frozen tumour and normal material. Short insert DNA libraries were prepared with the TruSeq DNA PCRfree sample preparation kit (Illumina) for paired-end sequencing at a minimum read length of 2 × 100 bp. Human DNA libraries were sequenced with...
Whole-exome sequencing
reagent used in the protocol.
- Use
- Whole-exome sequencing was performed on fresh-frozen tissue specimen from mice. The enrichment for the exome was performed with the SureSelectXT Mouse All Exon kit (Agilent) following the protocol of the manufacturer. The exon-enriched libraries were subjected to paired-end sequencing with a read-length of 2 ×...
Targeted enrichment sequencing
reagent used in the protocol.
- Use
- Targeted enrichment sequencing was performed on human FFPE and fresh-frozen tumour and normal specimen for the purpose of validating genome alterations in an independent cohort. The custom probe design was constructed with SureDesign (Agilent Technologies) enriching for the exons of 22 genes of interest. DNA librari...
DNA and RNA extractions
DNA was extracted from fresh-frozen tissues, EDTA blood, or FFPE samples using the Gentra Puregene DNA extraction kit (Qiagen) following the protocol of the manufacturer. DNA isolates were hydrated in TE-buffer and confirmed to be of high molecular weight (>10 kb) by agarose gel electrophoresis. Genomic DNA from fre...
- Use
- DNA was extracted from fresh-frozen tissues, EDTA blood, or FFPE samples using the Gentra Puregene DNA extraction kit (Qiagen) following the protocol of the manufacturer. DNA isolates were hydrated in TE-buffer and confirmed to be of high molecular weight (>10 kb) by agarose gel electrophoresis. Genomic DNA from fre...
DNA and RNA extractions
For RNA extractions, tissue sections were first lysed and homogenized with the Tissue Lyzer (Qiagen). Subsequent RNA extractions were performed with the Qiagen RNAeasy Mini Kit according to the instructions of the manufacturer. The RNA quality was assessed at the Bioanalyzer 2100 DNA Chip 7500 (Agilent Technologies)...
- Use
- For RNA extractions, tissue sections were first lysed and homogenized with the Tissue Lyzer (Qiagen). Subsequent RNA extractions were performed with the Qiagen RNAeasy Mini Kit according to the instructions of the manufacturer. The RNA quality was assessed at the Bioanalyzer 2100 DNA Chip 7500 (Agilent Technologies)...
Next-generation sequencing
All sequencing reactions were performed on an Illumina HiSeq 2000 instrument (Illumina, San Diego, CA, USA).
- Use
- All sequencing reactions were performed on an Illumina HiSeq 2000 instrument (Illumina, San Diego, CA, USA).
EdU incorporation assay
Transfected cells were treated with 10 µM EdU (5-ethynyl-2′-deoxyuridine) (Life Technologies) for 3 h before trypsinization for FACS. 1 × 10 5 live, GFP + cells were sorted and labelled with EdU using the Click-iT EdU Pacific Blue flow cytometry assay kit (Life Technologies). Cells were then run thro...
- Use
- Transfected cells were treated with 10 µM EdU (5-ethynyl-2′-deoxyuridine) (Life Technologies) for 3 h before trypsinization for FACS. 1 × 10 5 live, GFP + cells were sorted and labelled with EdU using the Click-iT EdU Pacific Blue flow cytometry assay kit (Life Technologies). Cells were then run thro...
Samples and clinical data
We collected 152 fresh-frozen clinical tumour specimens obtained from patients diagnosed with stage I-IV SCLC under institutional review board approval ( and ). The tumour samples were enriched for earlier stages and consisted of primary lung ( n = 148) and metastatic tumours ( n = 4) obtained by surgical rese...
- Use
- We collected 152 fresh-frozen clinical tumour specimens obtained from patients diagnosed with stage I-IV SCLC under institutional review board approval ( and ). The tumour samples were enriched for earlier stages and consisted of primary lung ( n = 148) and metastatic tumours ( n = 4) obtained by surgical rese...
Mouse SCLC models and tumour samples
SCLC tumours expressing the activated intracellular domain (ICD) of Notch1 (Notch1 ICD, N1ICD) and Notch2 (Notch2 ICD, N2ICD) were analysed in mouse models. Rosa26 Lox-stop-Lox-Notch1ICD ( LSL-N1ICD ) or Rosa26 Lox-stop-Lox-Notch2ICD ( LSL-N2ICD ) mice were obtained from Spyros Artavanis-Tsakonas and Exelixis. These...
- Use
- SCLC tumours expressing the activated intracellular domain (ICD) of Notch1 (Notch1 ICD, N1ICD) and Notch2 (Notch2 ICD, N2ICD) were analysed in mouse models. Rosa26 Lox-stop-Lox-Notch1ICD ( LSL-N1ICD ) or Rosa26 Lox-stop-Lox-Notch2ICD ( LSL-N2ICD ) mice were obtained from Spyros Artavanis-Tsakonas and Exelixis. These...
Universal inactivation of TP53 and RB1
Inactivating mutations in TP53 and RB1 have been shown to affect up to 90% and up to 65% of SCLC, respectively -. By contrast, our whole-genome sequencing analyses revealed that both genes were altered in all but two cases that exhibited signs of chromothripsis ( and ). TP53 and RB1 alterations were mostly in...
- Use
- Inactivating mutations in TP53 and RB1 have been shown to affect up to 90% and up to 65% of SCLC, respectively -. By contrast, our whole-genome sequencing analyses revealed that both genes were altered in all but two cases that exhibited signs of chromothripsis ( and ). TP53 and RB1 alterations were mostly in...
Universal inactivation of TP53 and RB1
The two tumours affected by chromothripsis displayed a similar pattern of massive genomic rearrangements between chromosomes 3 and 11 ( and ), but lacked shared fusion transcripts in the transcriptome sequencing data, suggesting that a particular fusion is not a common target ( and ). Of the genes on chromosomes 3 a...
- Use
- The two tumours affected by chromothripsis displayed a similar pattern of massive genomic rearrangements between chromosomes 3 and 11 ( and ), but lacked shared fusion transcripts in the transcriptome sequencing data, suggesting that a particular fusion is not a common target ( and ). Of the genes on chromosomes 3 a...
Mouse SCLC models and tumour samples
Software used for acquisition, scoring, statistics, or reporting.
- Use
- SCLC tumours expressing the activated intracellular domain (ICD) of Notch1 (Notch1 ICD, N1ICD) and Notch2 (Notch2 ICD, N2ICD) were analysed in mouse models. Rosa26 Lox-stop-Lox-Notch1ICD ( LSL-N1ICD ) or Rosa26 Lox-stop-Lox-Notch2ICD ( LSL-N2ICD ) mice were obtained from Spyros Artavanis-Tsakonas and Exelixis. These...
Differential expression for outlier studies
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Differential gene expression analysis was performed to compare the transcriptional profile of the two chromothripsis cases ( S02297 and S02353) with other non-chromothripsis SCLC cases and to thus identify outliers in the expression profile. The expression was analysed by computing z -scores for all samples referrin...
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Open quote workflowStep-by-step procedure
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EdU incorporation assay
Transfected cells were treated with 10 µM EdU (5-ethynyl-2′-deoxyuridine) (Life Technologies) for 3 h before trypsinization for FACS. 1 × 10 5 live, GFP + cells were sorted and labelled with EdU using the Click-iT EdU Pacific Blue flow cytometry assay kit (Life Technologies). Cells were then run through the BD FACSAria to analyse for per cent EdU incorporation.
DNA and RNA extractions
Nucleic acids were extracted from fresh-frozen tissue specimen which were processed to 15-30 sections each of 20 µm thickness at a cryostat maintaining a temperature of -20 °C (Leica). In the case of FFPE samples, 6-10 sections of 10 µm thickness were prepared.
Extended Data
a, Survival analysis of SCLC patients based on clinical stage and treatment options (surgery and/or chemotherapy). Statistical significance was determined by log-rank test. b, Analyses of clinical stage and smoking status and the respective effect on number and type of mutations, as well as mutational subclonality in tumours. Statistical significance was determined by Kruskal-Wallis analysis.
METHODS
Clinical correlation studies were performed with the study cohort of 110 SCLC patients considering age of diagnosis, gender, tumour stage, surgery, treatment with chemotherapeutics, smoking status, smoking history and overall survival ( and and ). The median follow-up time for this cohort of 110 SCLC patients was 69 months, and 31% of the patients were alive at the time of last follow-up ( and ). Smoking status was available for 88% ( n = 97) of the patients; 63% ( n = 69) reported a smoking history amounting to a median of 45 pack-years. Patients with a known smoking history were further subcategorized to heavy smokers (>30 pack-years), average smokers (10-30 pack-years) and light/never smokers (<10 pack-years).
Copy number analysis by Affymetrix SNP 6.0 arrays
Human DNA extracted from fresh-frozen tumour specimen was hybridized to Affymetrix Genome-Wide Human SNP array 6.0 following the manufacturer's instructions. The signal intensities were processed to analyse for chromosomal gene copy number data. Raw copy number signals and segmented copy number data were computed following the procedure described previously.
Copy number analysis by Affymetrix SNP 6.0 arrays
The raw, unsegmented copy number signals were used to analyse for significant copy number alterations applying the method CGARS. Significant amplifications were determined with the upper quantiles 0.25, 0.15, 0.1, and 0.05; deletions were computed in reference to the 0.25 lower quantile. The significance threshold was set at a q -value of 0.05 ( ).
Analysis of subclonal architecture
To determine the subclonal architecture from genome sequencing data, we first computed the cancer cell fraction (CCF; that is, the fraction of cancer cells carrying a particular mutation) of each called somatic point mutation. To this end, we first estimated the tumour purity, absolute copy numbers, and subclonal copy number changes using our previously described method and computed for each mutation the expected allelic fraction under clonality assumption. The quotient between the observed allelic fraction of a mutation with its corresponding expected allelic fraction then yields the CCF. To assess the clonal and subclonal populations we next identified distinct clusters in the CCF profile and assigned each mutation to the cluster of highest probability. In order to provide a measure for the subclonal architecture, we proposed the following score: Subclonality score = ∑ i = 1 n...
Analysis of subclonal architecture
As a low sequencing depth limits the robust identification of subclonal populations, we computed the genome-wide average contribution of a single mutated read to the CCF. For a given tumour purity p, average ploidy π, and mean coverage c, this measure is given by: Average increase of C C F per read = 2 ( 1 - p ) + p π p c The smaller the average increase of CCF per read, the more accurately the subclonality score can be determined since more subclonal mutations can be called from the sequencing data. In this study, the most limiting factor for assessing the subclonal diversity is the relatively low sequencing depth (35 × on average). We therefore used this measure to select the samples that are suitable for a reliable calculation of the subclonality score. To this end, we systematically scanned from the average increase of CCF per read form large to s...
Measurement outputs
What raw and processed outputs should exist?
We collected 152 fresh-frozen clinical tumour specimens obtained from patients diagnosed with stage I-IV SCLC under institutional review board approval ( and ). The tumour...
- 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
In order to assess the amount of genetic heterogeneity of SCLC, we developed a subclonality score, which can be interpreted as the probability that an arbitrary point mutation i...
- 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
Of the genes with an established role in murine SCLC (IV), we confirmed PTEN,. RBL1 and RBL2, which are closely related to RB1 (ref. ), similarly exhibited inactivating trans...
- 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
By analysing somatic copy number alterations, we confirmed previously known genomic losses within 3p pointing to focal events on 3p14.3-3p14.2 (harbouring FHIT ) and 3p12....
- 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 raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
No statistical methods were used to predetermine sample size.
from paperScoring or quantification
Quantify the primary readouts for this experiment: We collected 152 fresh-frozen clinical tumour specimens obtained from patients diagnosed with stage I-IV SCLC under institutional review board approval ( and ). The tumour...; In order to assess the amount of genetic heterogeneity of SCLC, we developed a subclonality score, which can be interpreted as the probability that an arbitrary point mutation i...; Of the genes with an established role in murine SCLC (IV), we confirmed PTEN,. RBL1 and RBL2, which are closely related to RB1 (ref. ), similarly exhibited inactivating trans...; By analysing somatic copy number alterations, we confirmed previously known genomic losses within 3p pointing to focal events on 3p14.3-3p14.2 (harbouring FHIT ) and 3p12.....
from paperStatistical comparison
No statistical methods were used to predetermine sample size.; SCLC tumours expressing the activated intracellular domain (ICD) of Notch1 (Notch1 ICD, N1ICD) and Notch2 (Notch2 ICD, N2ICD) were analysed in mouse models. Rosa26 Lox-stop-Lox-...; Locally clustered mutations (II) are indicative of functional selection ( P < 0.05,, Methods),. Of all genes, lists those alterations that occurred in more than 8% of the sam...; Across these five categories, mutations in CREBBP, EP300, TP73, RBL1, RBL2 and NOTCH family genes were largely mutually exclusive ( ), suggesting that they may exert similar pro...
from paperReporting output
Report representative outputs alongside summary comparisons for We collected 152 fresh-frozen clinical tumour specimens obtained from patients diagnosed with stage I-IV SCLC under institutional review board approval ( and ). The tumour..., In order to assess the amount of genetic heterogeneity of SCLC, we developed a subclonality score, which can be interpreted as the probability that an arbitrary point mutation i..., Of the genes with an established role in murine SCLC (IV), we confirmed PTEN,. RBL1 and RBL2, which are closely related to RB1 (ref. ), similarly exhibited inactivating trans..., By analysing somatic copy number alterations, we confirmed previously known genomic losses within 3p pointing to focal events on 3p14.3-3p14.2 (harbouring FHIT ) and 3p12.....
inferred from protocolStructured statistical methods
No statistical methods were used to predetermine sample size.; SCLC tumours expressing the activated intracellular domain (ICD) of Notch1 (Notch1 ICD, N1ICD) and Notch2 (Notch2 ICD, N2ICD) were analysed in mouse models. Rosa26 Lox-stop-Lox-...; Locally clustered mutations (II) are indicative of functional selection ( P < 0.05,, Methods),. Of all genes, lists those alterations that occurred in more than 8% of the sam...; Across these five categories, mutations in CREBBP, EP300, TP73, RBL1, RBL2 and NOTCH family genes were largely mutually exclusive ( ), suggesting that they may exert similar pro...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Transfected cells were treated with 10 µM EdU (5-ethynyl-2′-deoxyuridine) (Life Technologies) for 3 h before trypsinization for FACS. 1 × 10 5 live, GFP + cells were sorted and labelled with EdU using the Click-iT EdU Pacific Blue flow cytometry assay kit (Life Technologies). Cells were then run through the BD FACSAria to analyse for per cent EdU incorporation.
Nucleic acids were extracted from fresh-frozen tissue specimen which were processed to 15-30 sections each of 20 µm thickness at a cryostat maintaining a temperature of -20 °C (Leica). In the case of FFPE samples, 6-10 sections of 10 µm thickness were prepared.
a, Survival analysis of SCLC patients based on clinical stage and treatment options (surgery and/or chemotherapy). Statistical significance was determined by log-rank test. b, Analyses of clinical stage and smoking status and the respective effect on number and type of mutations, as well as mutational subclonality in tumours. Statistical significance was determined by Kruskal-Wallis analysis.
Clinical correlation studies were performed with the study cohort of 110 SCLC patients considering age of diagnosis, gender, tumour stage, surgery, treatment with chemotherapeutics, smoking status, smoking history and overall survival ( and and ). The median follow-up time for this cohort of 110 SCLC patients was 69 months, and 31% of the patients were alive at the time of last follow-up ( and ). Smoking status was available for 88% ( n = 97) of the patients; 63% ( n = 69) reported a smoking history amounting to a median of 45 pack-years. Patients with a known smoking history were further subcategorized to heavy smokers (>30 pack-years), average smokers (10-30 pack-years) and light/never smokers (<10 pack-years).
Human DNA extracted from fresh-frozen tumour specimen was hybridized to Affymetrix Genome-Wide Human SNP array 6.0 following the manufacturer's instructions. The signal intensities were processed to analyse for chromosomal gene copy number data. Raw copy number signals and segmented copy number data were computed following the procedure described previously.
The raw, unsegmented copy number signals were used to analyse for significant copy number alterations applying the method CGARS. Significant amplifications were determined with the upper quantiles 0.25, 0.15, 0.1, and 0.05; deletions were computed in reference to the 0.25 lower quantile. The significance threshold was set at a q -value of 0.05 ( ).
To determine the subclonal architecture from genome sequencing data, we first computed the cancer cell fraction (CCF; that is, the fraction of cancer cells carrying a particular mutation) of each called somatic point mutation. To this end, we first estimated the tumour purity, absolute copy numbers, and subclonal copy number changes using our previously described method and computed for each mutation the expected allelic fraction under clonality assumption. The quotient between the observed allelic fraction of a mutation with its corresponding expected allelic fraction then yields the CCF. To assess the clonal and subclonal populations we next identified distinct clusters in the CCF profile and assigned each mutation to the cluster of highest probability. In order to provide a measure for the subclonal architecture, we proposed the following score: Subclonality score = ∑ i = 1 n c ϕ i m i ∑ i = 0 n c ϕ i m i where i = 0 represents the clonal population, i = 1,…, nc the subclonal populations; φ i is the CCF of each population (thus, φ 0 ≈ 1), and m i is the number of mutations assigned to cluster i. This subclonality score can be inter...
As a low sequencing depth limits the robust identification of subclonal populations, we computed the genome-wide average contribution of a single mutated read to the CCF. For a given tumour purity p, average ploidy π, and mean coverage c, this measure is given by: Average increase of C C F per read = 2 ( 1 - p ) + p π p c The smaller the average increase of CCF per read, the more accurately the subclonality score can be determined since more subclonal mutations can be called from the sequencing data. In this study, the most limiting factor for assessing the subclonal diversity is the relatively low sequencing depth (35 × on average). We therefore used this measure to select the samples that are suitable for a reliable calculation of the subclonality score. To this end, we systematically scanned from the average increase of CCF per read form large to small values and detected the point of the most prominent change in the distribution of the subclonality score ( ).
Machine-readable layer
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"text": "Clinical correlation studies were performed with the study cohort of 110 SCLC patients considering age of diagnosis, gender, tumour stage, surgery, treatment with chemotherapeutics, smoking status, smoking history and overall survival ( and and ). The median follow-up time for this cohort of 110 SCLC patients was 69 months, and 31% of the patients were alive at the time of last follow-up ( and ). Smoking status was available for 88% ( n = 97) of the patients; 63% ( n = 69) reported a smoking history amounting to a median of 45 pack-years. Patients with a known smoking history were further subcategorized to heavy smokers (>30 pack-years), average smokers (10-30 pack-years) and light/never smokers (<10 pack-years)."
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"text": "To determine the subclonal architecture from genome sequencing data, we first computed the cancer cell fraction (CCF; that is, the fraction of cancer cells carrying a particular mutation) of each called somatic point mutation. To this end, we first estimated the tumour purity, absolute copy numbers, and subclonal copy number changes using our previously described method and computed for each mutation the expected allelic fraction under clonality assumption. The quotient between the observed allelic fraction of a mutation with its corresponding expected allelic fraction then yields the CCF. To assess the clonal and subclonal populations we next identified distinct clusters in the CCF profile and assigned each mutation to the cluster of highest probability. In order to provide a measure for the subclonal architecture, we proposed the following score: Subclonality score = ∑ i = 1 n..."
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"text": "As a low sequencing depth limits the robust identification of subclonal populations, we computed the genome-wide average contribution of a single mutated read to the CCF. For a given tumour purity p, average ploidy π, and mean coverage c, this measure is given by: Average increase of C C F per read = 2 ( 1 - p ) + p π p c The smaller the average increase of CCF per read, the more accurately the subclonality score can be determined since more subclonal mutations can be called from the sequencing data. In this study, the most limiting factor for assessing the subclonal diversity is the relatively low sequencing depth (35 × on average). We therefore used this measure to select the samples that are suitable for a reliable calculation of the subclonality score. To this end, we systematically scanned from the average increase of CCF per read form large to s..."
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