NEXT GENERATION SEQUENCING (NGS) ION TORRENT AMPLISEQ-TM TECHNOLOGY FOR THE IDENTIFICATION OF TP53 MUTATIONS ?SCREENING OF 39 CASES OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
(Abstract release date: 05/19/16)
EHA Library. De Stefano L. 06/09/16; 134675; PB1775
Dr. Laura De Stefano
Contributions
Contributions
Abstract
Abstract: PB1775
Type: Publication Only
Background
NGS technology allows to study the molecular genetics of CLL through the comprehensive detection of genetic lesions.
Aims
We describe the application of a NGS panel based on Ion AmpliSeqTM technology to analyze the full coding region of TP53 (ex 2–11) in a single workflow.
Methods
Purified B-cells, from 39 therapy-free CLL patients (pts) visiting our outpatient clinic. Library preparation and sequencing were performed on the Ion TorrentTM-PGM platform (Thermofisher) according to the manufacturer’s protocols. For each run, 10 pooled samples were loaded on a 316chip and sequenced (flow rate 500x). Data processing, filtering and base calling were performed using the Ion Torrent server v4.4.2. Based on Variant Caller Parameters we accepted all variants with an allele frequency (VAF)>5% with high quality, rejected variants had VAF<3% and low quality. Genomic sequences were compared to the IARC TP53 database and confirmed using Sanger sequencing.
Results
ION-PGM sequencing data showed a high coverage in 90% of sequenced amplicons with average coverage uniformity of 89%, average mean depth of 13130 (range 8812–17960), and 97% of mapped reads on target. We identified 84 variants in 39 pts (including synonymous [N=2/2 pts] and non-synonymous [NS] mutations [n=44/26 pts]) in TP53 regions. A total of 84 variants were identified in 30/39pts (77%). Overall, of the 39 pts analyzed, 9 pts were TP53WT, while 30/39 76.9% presented variants along the entire TP53 sequence. 3/39 presented only intronic mutations and were considered TP53WT; 1/39 pts with a 3’UTR mutation with VAF=4.5%. In the remaining 26 pts NGS identified 46 exonic mutations (range, 1-5 variants/patient): 6/26 with clonal mutations only, defined as VAF>10%, (23.1%); 16/26 (61.5%) clonal and subclonal; and in 4/26 (15.4%) only subclonal variations were identified. Exon mutation hotspots were located in ex-7 (domain IV), ex-11 (C-terminal) and the 3’UTR, with 13, 11, and 10 variants, respectively. Overall, a notable % of mutations were identified having a VAF between 3–5%(60/84 variants, 71.4%) and between 5–10% (6/84, 5.9%), which may be considered subclonal variants defined as below the detection limit of Sanger sequencing. Of the 46 exon variants (26 pts) identified, 44 were NS mutations with the following effects at the protein level: 28 deleterious, 3 frameshift, 12 neutral, 1 stop/loss and 2 unclassified mutations and were indicated as alterations likely producing nonfunctional protein according to in silico analysis. The mutations in the 3’UTR (10) and 5’UTR (1) could potentially affect regulation of TP53 gene expression by target miRNA and/or transcription factors. Correlation with FISH analysis showed that of the 4 pts with del17p, 3 pts presented a clonal TP53 mutation (with VAF range 22.5–94.7%) in the remaining allele, while the 4th presented a subclonal mutation with a VAF of 3.1%, of note this was the only del17p patient alive in this cohort.
Conclusion
The ION PGM-TP53 panel offers an easy to use platform for the evaluation of small clonal TP53 mutations. A total of 67/84mutations were detected as small TP53-mutant subclones having a VAF<10%, that would likely be considered WT by Sanger sequencing, demonstrating the high sensitivity of the technique. The presence of subclonal mutations could anticipate the development of a chemo-refractory phenotype among CLL pts requiring treatment. We are currently evaluating TP53 clonal alterations identified by NGS screening in these CLL pts prospectively. Special thanks to Celegene and AIRC-CARICALRegional Grant 16695.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Clonal expansion, Mutation
Type: Publication Only
Background
NGS technology allows to study the molecular genetics of CLL through the comprehensive detection of genetic lesions.
Aims
We describe the application of a NGS panel based on Ion AmpliSeqTM technology to analyze the full coding region of TP53 (ex 2–11) in a single workflow.
Methods
Purified B-cells, from 39 therapy-free CLL patients (pts) visiting our outpatient clinic. Library preparation and sequencing were performed on the Ion TorrentTM-PGM platform (Thermofisher) according to the manufacturer’s protocols. For each run, 10 pooled samples were loaded on a 316chip and sequenced (flow rate 500x). Data processing, filtering and base calling were performed using the Ion Torrent server v4.4.2. Based on Variant Caller Parameters we accepted all variants with an allele frequency (VAF)>5% with high quality, rejected variants had VAF<3% and low quality. Genomic sequences were compared to the IARC TP53 database and confirmed using Sanger sequencing.
Results
ION-PGM sequencing data showed a high coverage in 90% of sequenced amplicons with average coverage uniformity of 89%, average mean depth of 13130 (range 8812–17960), and 97% of mapped reads on target. We identified 84 variants in 39 pts (including synonymous [N=2/2 pts] and non-synonymous [NS] mutations [n=44/26 pts]) in TP53 regions. A total of 84 variants were identified in 30/39pts (77%). Overall, of the 39 pts analyzed, 9 pts were TP53WT, while 30/39 76.9% presented variants along the entire TP53 sequence. 3/39 presented only intronic mutations and were considered TP53WT; 1/39 pts with a 3’UTR mutation with VAF=4.5%. In the remaining 26 pts NGS identified 46 exonic mutations (range, 1-5 variants/patient): 6/26 with clonal mutations only, defined as VAF>10%, (23.1%); 16/26 (61.5%) clonal and subclonal; and in 4/26 (15.4%) only subclonal variations were identified. Exon mutation hotspots were located in ex-7 (domain IV), ex-11 (C-terminal) and the 3’UTR, with 13, 11, and 10 variants, respectively. Overall, a notable % of mutations were identified having a VAF between 3–5%(60/84 variants, 71.4%) and between 5–10% (6/84, 5.9%), which may be considered subclonal variants defined as below the detection limit of Sanger sequencing. Of the 46 exon variants (26 pts) identified, 44 were NS mutations with the following effects at the protein level: 28 deleterious, 3 frameshift, 12 neutral, 1 stop/loss and 2 unclassified mutations and were indicated as alterations likely producing nonfunctional protein according to in silico analysis. The mutations in the 3’UTR (10) and 5’UTR (1) could potentially affect regulation of TP53 gene expression by target miRNA and/or transcription factors. Correlation with FISH analysis showed that of the 4 pts with del17p, 3 pts presented a clonal TP53 mutation (with VAF range 22.5–94.7%) in the remaining allele, while the 4th presented a subclonal mutation with a VAF of 3.1%, of note this was the only del17p patient alive in this cohort.
Conclusion
The ION PGM-TP53 panel offers an easy to use platform for the evaluation of small clonal TP53 mutations. A total of 67/84mutations were detected as small TP53-mutant subclones having a VAF<10%, that would likely be considered WT by Sanger sequencing, demonstrating the high sensitivity of the technique. The presence of subclonal mutations could anticipate the development of a chemo-refractory phenotype among CLL pts requiring treatment. We are currently evaluating TP53 clonal alterations identified by NGS screening in these CLL pts prospectively. Special thanks to Celegene and AIRC-CARICALRegional Grant 16695.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Clonal expansion, Mutation
Abstract: PB1775
Type: Publication Only
Background
NGS technology allows to study the molecular genetics of CLL through the comprehensive detection of genetic lesions.
Aims
We describe the application of a NGS panel based on Ion AmpliSeqTM technology to analyze the full coding region of TP53 (ex 2–11) in a single workflow.
Methods
Purified B-cells, from 39 therapy-free CLL patients (pts) visiting our outpatient clinic. Library preparation and sequencing were performed on the Ion TorrentTM-PGM platform (Thermofisher) according to the manufacturer’s protocols. For each run, 10 pooled samples were loaded on a 316chip and sequenced (flow rate 500x). Data processing, filtering and base calling were performed using the Ion Torrent server v4.4.2. Based on Variant Caller Parameters we accepted all variants with an allele frequency (VAF)>5% with high quality, rejected variants had VAF<3% and low quality. Genomic sequences were compared to the IARC TP53 database and confirmed using Sanger sequencing.
Results
ION-PGM sequencing data showed a high coverage in 90% of sequenced amplicons with average coverage uniformity of 89%, average mean depth of 13130 (range 8812–17960), and 97% of mapped reads on target. We identified 84 variants in 39 pts (including synonymous [N=2/2 pts] and non-synonymous [NS] mutations [n=44/26 pts]) in TP53 regions. A total of 84 variants were identified in 30/39pts (77%). Overall, of the 39 pts analyzed, 9 pts were TP53WT, while 30/39 76.9% presented variants along the entire TP53 sequence. 3/39 presented only intronic mutations and were considered TP53WT; 1/39 pts with a 3’UTR mutation with VAF=4.5%. In the remaining 26 pts NGS identified 46 exonic mutations (range, 1-5 variants/patient): 6/26 with clonal mutations only, defined as VAF>10%, (23.1%); 16/26 (61.5%) clonal and subclonal; and in 4/26 (15.4%) only subclonal variations were identified. Exon mutation hotspots were located in ex-7 (domain IV), ex-11 (C-terminal) and the 3’UTR, with 13, 11, and 10 variants, respectively. Overall, a notable % of mutations were identified having a VAF between 3–5%(60/84 variants, 71.4%) and between 5–10% (6/84, 5.9%), which may be considered subclonal variants defined as below the detection limit of Sanger sequencing. Of the 46 exon variants (26 pts) identified, 44 were NS mutations with the following effects at the protein level: 28 deleterious, 3 frameshift, 12 neutral, 1 stop/loss and 2 unclassified mutations and were indicated as alterations likely producing nonfunctional protein according to in silico analysis. The mutations in the 3’UTR (10) and 5’UTR (1) could potentially affect regulation of TP53 gene expression by target miRNA and/or transcription factors. Correlation with FISH analysis showed that of the 4 pts with del17p, 3 pts presented a clonal TP53 mutation (with VAF range 22.5–94.7%) in the remaining allele, while the 4th presented a subclonal mutation with a VAF of 3.1%, of note this was the only del17p patient alive in this cohort.
Conclusion
The ION PGM-TP53 panel offers an easy to use platform for the evaluation of small clonal TP53 mutations. A total of 67/84mutations were detected as small TP53-mutant subclones having a VAF<10%, that would likely be considered WT by Sanger sequencing, demonstrating the high sensitivity of the technique. The presence of subclonal mutations could anticipate the development of a chemo-refractory phenotype among CLL pts requiring treatment. We are currently evaluating TP53 clonal alterations identified by NGS screening in these CLL pts prospectively. Special thanks to Celegene and AIRC-CARICALRegional Grant 16695.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Clonal expansion, Mutation
Type: Publication Only
Background
NGS technology allows to study the molecular genetics of CLL through the comprehensive detection of genetic lesions.
Aims
We describe the application of a NGS panel based on Ion AmpliSeqTM technology to analyze the full coding region of TP53 (ex 2–11) in a single workflow.
Methods
Purified B-cells, from 39 therapy-free CLL patients (pts) visiting our outpatient clinic. Library preparation and sequencing were performed on the Ion TorrentTM-PGM platform (Thermofisher) according to the manufacturer’s protocols. For each run, 10 pooled samples were loaded on a 316chip and sequenced (flow rate 500x). Data processing, filtering and base calling were performed using the Ion Torrent server v4.4.2. Based on Variant Caller Parameters we accepted all variants with an allele frequency (VAF)>5% with high quality, rejected variants had VAF<3% and low quality. Genomic sequences were compared to the IARC TP53 database and confirmed using Sanger sequencing.
Results
ION-PGM sequencing data showed a high coverage in 90% of sequenced amplicons with average coverage uniformity of 89%, average mean depth of 13130 (range 8812–17960), and 97% of mapped reads on target. We identified 84 variants in 39 pts (including synonymous [N=2/2 pts] and non-synonymous [NS] mutations [n=44/26 pts]) in TP53 regions. A total of 84 variants were identified in 30/39pts (77%). Overall, of the 39 pts analyzed, 9 pts were TP53WT, while 30/39 76.9% presented variants along the entire TP53 sequence. 3/39 presented only intronic mutations and were considered TP53WT; 1/39 pts with a 3’UTR mutation with VAF=4.5%. In the remaining 26 pts NGS identified 46 exonic mutations (range, 1-5 variants/patient): 6/26 with clonal mutations only, defined as VAF>10%, (23.1%); 16/26 (61.5%) clonal and subclonal; and in 4/26 (15.4%) only subclonal variations were identified. Exon mutation hotspots were located in ex-7 (domain IV), ex-11 (C-terminal) and the 3’UTR, with 13, 11, and 10 variants, respectively. Overall, a notable % of mutations were identified having a VAF between 3–5%(60/84 variants, 71.4%) and between 5–10% (6/84, 5.9%), which may be considered subclonal variants defined as below the detection limit of Sanger sequencing. Of the 46 exon variants (26 pts) identified, 44 were NS mutations with the following effects at the protein level: 28 deleterious, 3 frameshift, 12 neutral, 1 stop/loss and 2 unclassified mutations and were indicated as alterations likely producing nonfunctional protein according to in silico analysis. The mutations in the 3’UTR (10) and 5’UTR (1) could potentially affect regulation of TP53 gene expression by target miRNA and/or transcription factors. Correlation with FISH analysis showed that of the 4 pts with del17p, 3 pts presented a clonal TP53 mutation (with VAF range 22.5–94.7%) in the remaining allele, while the 4th presented a subclonal mutation with a VAF of 3.1%, of note this was the only del17p patient alive in this cohort.
Conclusion
The ION PGM-TP53 panel offers an easy to use platform for the evaluation of small clonal TP53 mutations. A total of 67/84mutations were detected as small TP53-mutant subclones having a VAF<10%, that would likely be considered WT by Sanger sequencing, demonstrating the high sensitivity of the technique. The presence of subclonal mutations could anticipate the development of a chemo-refractory phenotype among CLL pts requiring treatment. We are currently evaluating TP53 clonal alterations identified by NGS screening in these CLL pts prospectively. Special thanks to Celegene and AIRC-CARICALRegional Grant 16695.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Clonal expansion, Mutation
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