Abstract #313 Development of a single-step isolation ... · PDF fileDevelopment of a...
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Development of a single-step isolation platform to analyze exosomal RNA and cell-free DNA in plasma from cancer patients D. Enderle 1 , T. Koestler 1 , A. Spiel 1 , K. Brinkmann 1 , S. Bentink 1 , J. Skog 2 , M. Noerholm 1 (1) Exosome Diagnostics GmbH, Munich, Germany. (2) Exosome Diagnostics Inc., Cambridge, MA, USA. Introduction and Methods: Circulating nucleic acids in the bloodstream of cancer patients are of great interest to medical research because of their potential to yield information on a patient’s disease status and guide treatment options without requiring a tissue biopsy. Any diagnostic test that seeks to utilize biofluids for mutation analysis needs a platform that can maximize the capture of tumour-derived mutations in circulation. Blood plasma contains at least two cell-free sources of nucleic acids: circulating cell-free DNA (cfDNA), generated from apoptotic or necrotic cells, and RNA enclosed in extracellular vesicles including exosomes (exoRNA), which are actively secreted by cells in the body. We devised a method to simultaneously isolate both exoRNA and cfDNA from blood plasma using a single isolation column, maximizing assay sensitivity and capturing molecular information from both biologically different sources. The isolation of high quality nucleic enables effective downstream processing by RT-qPCR and targeted re-sequencing with NGS. Here we utilized our EXO52 technology platform to co-isolate and analyze exoRNA and cfDNA in blood plasma samples from 16 metastatic colorectal cancer (mCRC) patients. Conclusions: • EXO52 technology platform provides a convenient and reproducible method for co-isolating all exoRNA and cfDNA from high volumes of biofluids. • Simultaneously analyzing both exoRNA and cfDNA substantially increases the molecules available for rare mutant detection by qPCR and NextGen Sequencing versus cfDNA alone. • EXO52 platform enables robust, highly sensitive detection of actionable mutations in plasma with important clinical utility. • Bind cfDNA and vesicles to EXO52 spin-column membrane & wash • On-column QIAzol lysis and release of RNA and DNA • Phenol/Chloroform extraction • Ethanol conditioning • Bind to silica column and wash • Elute DNA and RNA into small volume Figure 2. Co-isolation of exoRNA and cfDNA from biofluids using EXO52 technology platform. The EXO52 platform employs a proprietary capture mechanism in a disposable spin- column format to enable routine parallel co-extraction of exoRNA and cfDNA from biofluids. EXO52 spin-column Figure 5. exoRNA and cfDNA both contribute substantially to the total nucleic acids harvested from blood plasma. One milliliter plasma from 5 healthy donors were isolated either using the exoRNeasy kit (RNA) or an EXO52 isolation with an reverse transcription step (RNA+ DNA) or without (DNA). Absolute quantification by RT-qPCR is presented as a boxplot indicating the median copy number per mL plasma with individual donors plotted as individual shapes. copies / 1 mL plasma 0% 20% 40% 60% 80% 100% 120% % of PCR signal from 2 mL plasma isolation EXO52 RNA+DNA RNA in flowthrough DNA in flowthrough Figure 4. Sequential isolation of nucleic acids from 2 mL of blood plasma. Blood plasma from a normal healthy donor was passed through an EXO52 column, and the material left in the flowthrough was isolated using either QIAGEN’s “exoRNeasy” kit (RNA) or “circulating nucleic acids kit” (DNA). The overall yield is compared to EXO52 (RNA+DNA) using RT-qPCR against BRAF , KRAS and 18S genes using the delta CT method. Error bars represent three replicate sample isolations. EXO52 column binds all mRNA and DNA in plasma 18S BRAF KRAS PIK3CA Figure 1. Sources of extracellular RNA and DNA in circulation. Exosomes are actively released by living cells directly from the plasma membrane (A) or via the multivesicular body pathway (A’), carrying RNA into circulation (exoRNA). Dying cells may release cell-free DNA (cfDNA) in apoptotic vesicles (B) or as free-circulating nucleosomes (B’). Figure 3. Size distribution of nucleic acids in plasma. Complete nucleic acids isolated from 1 mL plasma were subjected to either RNase A digestion (“cfDNA”), DNase I digestion (“exoRNA”) or mock treatment (“EXO52”). After reaction cleanup the size distribution of nucleic acids present in the isolation were measured using an Agilent Bioanalyzer Pico 6000 assay. EXO52 exoRNA +cfDNA 16 ng / mL BRAF NRAS KRAS Abstract #313 Poster #093 Cosmic Muta,on Pa,ent No. DNA RNA+DNA FF Tissue K601E 3909 1 1 K601E 3923 5 7 L597V 3923 0 2 T599T 3923 0 6 V600E 3137 4 28 + W604* 3923 0 6 G12A 3133 0 0 + G12A 3923 0 4 G12C 3024 5 5 G12D 3015 0.9 6 G12D 3018 0.5 14 G12D 3137 0 0 + G12D 3142 0 0.7 + G12D 3900 0 9 G12D 3922 35 104 + G12V 3009 0 0 + G12V 3018 0 0 + G12V 3901 0 0.2 + G12V 3909 3795 5049 + G12V 3917 65 102 + G12V 3923 30157 36805 + G13D 3126 3 32 + Q22* 3137 0 2 Figure 8. Combining isolation and analysis of exoRNA and cfDNA increases detectable tumor mutations in plasma. BRAF and KRAS mutant copies detected in 3 mL plasma of 16 patients with mCRC. Samples were collected under IRB by a commercial CRO from patient with late stage mCRC (stage IV). Blood was collected prior to surgery and plasma processed immediately. EXO52 extractions were reverse transcribed (RNA+DNA) or left untreated (DNA). This table lists all mutations from all patients that had either a mutation present at ≥ 1 copy (dark cells) or a positive signal from a fresh frozen (FF) tissue. EGFR was assessed but not detected. All mutations found in cfDNA are detected with increased copy numbers in the sample using both exoRNA and cfDNA demonstrating the benefit of using the exoRNA fraction for rare mutant detection in plasma. Mutant Copies KRAS BRAF Isolation of exoRNA and cfDNA from plasma of patients with late-stage metastatic colorectal cancer (mCRC) EXO52 exoRNA and cfDNA acttgaagtccaaatct acttcaagtccaaatct acttcaagtccaaatct Mutant Copies I. Draw blood into plasma tubes, exclude cellular material using a 0.8 µm syringe filter and store aliquots of filtered plasma at -80°C. II. Co-isolate exoRNA and cfDNA from 3 mL of plasma using an EXO52 column. III. Reverse transcription to include the exoRNA fraction (RNA+DNA) and no RT control as a comparison (DNA). IV. Targeted enrichment of BRAF, NRAS, KRAS, PIK3CA, MEK1 and EGFR using a multiplex PCR panel for next generation sequencing (NGS). V. Sequencing on an Illumina MiSeq™ and absolute quantification of input material. VI. Bioinformatic analysis including noise correction and calculation of mutant copy numbers ; see fig. 7 and 8. Figure 7. EXO52 platform enables detection of more mutations in plasma samples of mCRC patients. 3 mL plasma from 16 patients with mCRC was processed and analyzed as detailed above. Mutations in BRAF, KRAS or EGFR with a minimum of ≥ 1 copy into the assay and listed in the COSMIC database of known somatic mutations were considered detected and plotted into a Venn diagram. Reverse transcription of the EXO52 eluate (exoRNA+cfDNA) enables the detection of an additional set of 8 mutations when compared to the untreated sample (cfDNA). 8 9 0 EXO52 exoRNA+cfDNA cfDNA 4873 apopto,c / necro,c cell living cell A A’ B B’ • Extracellular vesicles, mainly 50-200 nm in size, are actively secreted by the cell and carry a snapshot of the the body’s transcriptome (exoRNA) • Circulating cell-free DNA (cfDNA) is thought to exist mainly as free- circulating nucleosomes, shed by apoptotic or necrotic cells. cfDNA 3 ng / mL exoRNA 7 ng / mL © 2014 Exosome Diagnos8cs, Inc. This work was supported partly by the m4 spitzencluster initiative
Transcript of Abstract #313 Development of a single-step isolation ... · PDF fileDevelopment of a...
Development of a single-step isolation platform to analyze exosomal RNA and cell-free DNA in plasma from cancer patients
D. Enderle 1, T. Koestler 1, A. Spiel 1, K. Brinkmann 1, S. Bentink 1, J. Skog 2, M. Noerholm 1
(1) Exosome Diagnostics GmbH, Munich, Germany. (2) Exosome Diagnostics Inc., Cambridge, MA, USA.
Introduction and Methods: Circulating nucleic acids in the bloodstream of cancer patients are of great interest to medical research because of their potential to yield information on a patient’s disease status and guide treatment options without requiring a tissue biopsy. Any diagnostic test that seeks to utilize biofluids for mutation analysis needs a platform that can maximize the capture of tumour-derived mutations in circulation. Blood plasma contains at least two cell-free sources of nucleic acids: circulating cell-free DNA (cfDNA), generated from apoptotic or necrotic cells, and RNA enclosed in extracellular vesicles including exosomes (exoRNA), which are actively secreted by cells in the body. We devised a method to simultaneously isolate both exoRNA and cfDNA from blood plasma using a single isolation column, maximizing assay sensitivity and capturing molecular information from both biologically different sources. The isolation of high quality nucleic enables effective downstream processing by RT-qPCR and targeted re-sequencing with NGS. Here we utilized our EXO52 technology platform to co-isolate and analyze exoRNA and cfDNA in blood plasma samples from 16 metastatic colorectal cancer (mCRC) patients.
Conclusions: • EXO52 technology platform provides a convenient and
reproducible method for co-isolating all exoRNA and cfDNA from high volumes of biofluids.
• Simultaneously analyzing both exoRNA and cfDNA substantially increases the molecules available for rare mutant detection by qPCR and NextGen Sequencing versus cfDNA alone.
• EXO52 platform enables robust, highly sensitive detection of actionable mutations in plasma with important clinical utility.
• Bind cfDNA and vesicles to EXO52 spin-column membrane & wash
• On-column QIAzol lysis and release of RNA and DNA
• Phenol/Chloroform extraction • Ethanol conditioning • Bind to silica column and wash • Elute DNA and RNA into small volume
Figure 2. Co-isolation of exoRNA and cfDNA from biofluids using EXO52 technology platform. The EXO52 platform employs a proprietary capture mechanism in a disposable spin-column format to enable routine parallel co-extraction of exoRNA and cfDNA from biofluids.
EXO52 spin-column Figure 5. exoRNA and cfDNA both contribute substantially to the total nucleic acids harvested from blood plasma. One milliliter plasma from 5 healthy donors were isolated either using the exoRNeasy kit (RNA) or an EXO52 isolation with an reverse transcription step (RNA+ DNA) or without (DNA). Absolute quantification by RT-qPCR is presented as a boxplot indicating the median copy number per mL plasma with individual donors plotted as individual shapes.
copi
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asm
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80%
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f PC
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l fro
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EXO52 RNA+DNA
RNA in flowthrough
DNA in flowthrough
Figure 4. Sequential isolation of nucleic acids from 2 mL of blood plasma. Blood plasma from a normal healthy donor was passed through an EXO52 column, and the material left in the flowthrough was isolated using either QIAGEN’s “exoRNeasy” kit (RNA) or “circulating nucleic acids kit” (DNA). The overall yield is compared to EXO52 (RNA+DNA) using RT-qPCR against BRAF, KRAS and 18S genes using the delta CT method. Error bars represent three replicate sample isolations.
EXO52 column binds all mRNA and DNA in plasma
18S
BRAF
KRAS PIK3CA
Figure 1. Sources of extracellular RNA and DNA in circulation. Exosomes are actively released by living cells directly from the plasma membrane (A) or via the multivesicular body pathway (A’), carrying RNA into circulation (exoRNA). Dying cells may release cell-free DNA (cfDNA) in apoptotic vesicles (B) or as free-circulating nucleosomes (B’).
Figure 3. Size distribution of nucleic acids in plasma. Complete nucleic acids isolated from 1 mL plasma were subjected to either RNase A digestion (“cfDNA”), DNase I digestion (“exoRNA”) or mock treatment (“EXO52”). After reaction cleanup the size distribution of nucleic acids present in the isolation were measured using an Agilent Bioanalyzer Pico 6000 assay.
EXO52 exoRNA +cfDNA 16 ng / mL
BRAF NRAS KRAS
Abstract #313 Poster #093
Cosmic Muta,on Pa,ent No. DNA RNA+DNA FF Tissue
K601E 3909 1 1 -‐ K601E 3923 5 7 -‐ L597V 3923 0 2 -‐ T599T 3923 0 6 -‐ V600E 3137 4 28 + W604* 3923 0 6 -‐ G12A 3133 0 0 + G12A 3923 0 4 -‐ G12C 3024 5 5 -‐ G12D 3015 0.9 6 -‐ G12D 3018 0.5 14 -‐ G12D 3137 0 0 + G12D 3142 0 0.7 + G12D 3900 0 9 -‐ G12D 3922 35 104 + G12V 3009 0 0 + G12V 3018 0 0 + G12V 3901 0 0.2 + G12V 3909 3795 5049 + G12V 3917 65 102 + G12V 3923 30157 36805 + G13D 3126 3 32 + Q22* 3137 0 2 -‐
Figure 8. Combining isolation and analysis of exoRNA and cfDNA increases detectable tumor mutations in plasma. BRAF and KRAS mutant copies detected in 3 mL plasma of 16 patients with mCRC. Samples were collected under IRB by a commercial CRO from patient with late stage mCRC (stage IV). Blood was collected prior to surgery and plasma processed immediately. EXO52 extractions were reverse transcribed (RNA+DNA) or left untreated (DNA). This table lists all mutations from all patients that had either a mutation present at ≥ 1 copy (dark cells) or a positive signal from a fresh frozen (FF) tissue. EGFR was assessed but not detected. All mutations found in cfDNA are detected with increased copy numbers in the sample using both exoRNA and cfDNA demonstrating the benefit of using the exoRNA fraction for rare mutant detection in plasma.
Mutant Copies
KRAS
BRAF
Isolation of exoRNA and cfDNA from plasma of patients with late-stage metastatic colorectal cancer (mCRC)
EXO52 exoRNA and cfDNA
acttgaagtccaaatct acttcaagtccaaatct acttcaagtccaaatct
Mutant Copies
I. Draw blood into plasma tubes, exclude cellular material using a 0.8 µm syringe filter and store aliquots of filtered plasma at -80°C.
II. Co-isolate exoRNA and cfDNA from 3 mL of plasma using an EXO52 column.
III. Reverse transcription to include the exoRNA fraction (RNA+DNA) and no RT control as a comparison (DNA).
IV. Targeted enrichment of BRAF, NRAS, KRAS, PIK3CA, MEK1 and EGFR using a multiplex PCR panel for next generation sequencing (NGS).
V. Sequencing on an Illumina MiSeq™ and absolute quantification of input material.
VI. Bioinformatic analysis including noise correction and calculation of mutant copy numbers ; see fig. 7 and 8.
Figure 7. EXO52 platform enables detection of more mutations in plasma samples of mCRC patients. 3 mL plasma from 16 patients with mCRC was processed and analyzed as detailed above. Mutations in BRAF, KRAS or EGFR with a minimum of ≥ 1 copy into the assay and listed in the COSMIC database of known somatic mutations were considered detected and plotted into a Venn diagram. Reverse transcription of the EXO52 eluate (exoRNA+cfDNA) enables the detection of an additional set of 8 mutations when compared to the untreated sample (cfDNA).
8 9 0
EXO52 exoRNA+cfDNA
cfDNA
4873
apopto,c / necro,c cell
living cell
AA’
B
B’
• Extracellular vesicles, mainly 50-200 nm in size, are actively secreted by the cell and carry a snapshot of the the body’s transcriptome (exoRNA)
• Circulating cell-free DNA (cfDNA) is thought to exist mainly as free-circulating nucleosomes, shed by apoptotic or necrotic cells.
cfDNA
3 ng / mL
exoRNA
7 ng / mL
© 2014 Exosome Diagnos8cs, Inc.
This work was supported partly by the m4 spitzencluster initiative
