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ABSTRACTCurrently the standard practice in tumor biomarker research still relies on invasive tumor biopsy from local sites following by molecular testing or NGS assay. However, this only provides a very limited characterization of tumor composition. Recent studies in non-invasive biomarker research have demonstrated the potential advantages of using cell-free nucleic acids isolated from blood plasma to study genetic heterogeneity of tumor population and dissect the complex cancer clonal architecture. However, it has been challenging to apply to practical research due to low sample yield and sensitivity of detection approaches. Moreover, presence of both cfDNA and cfRNA requires methods capable of interrogating both types of analytes to maximize the utility of each plasma sample and characterize the comprehensive spectrum of mutations including single nucleotide variants, gene amplifications, and structural variants.

Recently developed Oncomine™ Pan-Cancer Cell-free Assay employs an amplification-based approach from Ion Torrent NGS technology and achieves exceptional sensitivity and specificity with input amount as little as 20 ng. It includes the most comprehensive genetic content to simultaneously interrogate both cfDNA and cfRNA. Multiple libraries were pooled together for templating on Ion Chef™ and sequenced on Ion GeneStudio S5 systems.

In this study, a panel of cancer cell lines harboring multiple variant types were selected and cultured for extended time after apoptosis. We were able to extract cell-free nucleic acids that were released into the cell media and mimic the circulating DNA profile of liquid biopsy samples using in vitro model. Using Oncomine™ Pan-Cancer Cell-free Assay, we successfully detected all the expected variants in these cancer cell lines including gene amplification (MET, ERBB2, CDK4) and fusion variants (ALK fusion and MET exon skipping). Subsequently, we applied this assay to a set of longitudinal liquid biopsy samples collected from a human subject with NSCLC during the course of 15 months. The results showed that a well-known TP53 mutation R248Q was consistently detected in the longitudinal samples with varying allelic frequencies. Interestingly, additional gene amplifications including MET, CDK4 and FGFR3 were identified at late time points. Furthermore, these observations were confirmed by digital PCR and concordant with FISH analyses in solid tumor. Overall, the study demonstrates that Pan-Cancer assay provides a unique and complete NGS solution for comprehensive genetic mutation assessment using in vitro and in vivo liquid biopsy models.

Ru Cao1, Kris Lea1, Jeff Schageman1, Khalid Hanif1, Yanchun Li1, Jian Gu1, Priyanka Kshatriya1, Andrea Luchetti1, Chris Allen1, Luca Quagliata1, Harriet Wikman2, Sonja Loges2, Kelli S. Bramlett11Thermo Fisher Scientific, 2130 Woodward Street, Austin, Texas 787442Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Characterization of Genetic Mutation Spectra and Identification of Gene Amplification and Fusion Variants in Cell-Free Nuclei Acid from Cultured Cancer Cell Media and Liquid Biopsy Specimens Using Oncomine™ Pan-Cancer Cell-Free Assay

CONCLUSIONS• OncomineTM Pan-Cancer cell-free assay provides the most efficient

NGS solution with the most comprehensive genetic content for oncology molecule profiling of liquid biopsy samples.

• Assay validation demonstrates unparalleled and reproducible analytical performance for each variant type including SNV, CNV (MET, ERBB2, EGFR, MYC, FGFR1, FGFR2, and CCND1), RNA fusion (ALK, ROS1, and RET) and MET exon skipping.

• All the expected copy number variants in a set of cancer cell lines were detected with fold changes comparable to the results measured by digital PCR.

• In the retrospective research study on a set of longitude liquid biopsy samples collected from male research subject with NSCLC, Pan-Cancer cell-free assay was able to detect several gene amplifications, showed concordance with FISH analyses in solid tumor, and subsequently confirmed by digital PCR analysis.

• MET amplification was identified at later stage and is most likely responsible for the failure of the second generation ALK inhibitors including Alectinib and Ceritinib.

RESULTS

Figure 1. OncomineTM Pan-Cancer Cell-free Assay Workflow

Figure 1. The OncomineTM Pan-Cancer cell-free assay enables a fast 3-day workflow that starts with cell-free nucleic acid purification from a single tube of blood and library preparation to interrogate both DNA and RNA in a single reaction. The amplified library will undergo high-throughput semiconductor sequencing, with results analyzed and reported through an integrated bioinformatics solution.

TRADEMARKS/LICENSINGFor Research Use Only. Not for use in diagnostic procedures.

© 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license

Table 1. OncomineTMPan-Cancer Cell-free Assay Content

Table 1. OncomineTM Pan-Cancer cell-free assay provides broad coverage across 52 unique genes that detects DNA and RNA variants across multiple cancer types. The workflow also offers multiplexing flexibility utilizing different chip configurations available to GeneStudio S5 systems to accommodate the needs for desired throughput..

Table 2. Assay reproducibility across Users, Instruments, and Reagent Lots for SNV Detection

Table 2. The reproducibility of SNV detection sensitivity and specificity were demonstrated across three users, four instruments, and two lots of Pan-Cancer panel using contrived controls generated by titrating AcroMetrixTM

oncology hotspot control in a genomic DNA as background at 0.1% and 0.5%, respectively.

User Average (n=2)

All Average (n=8)

User Average (n=2)

All Average (n=8)

1 99.1% 99.9%2 99.6% 100.0%3 99.1% 100.0%

2 1 99.6% 100.0%

1

SampleSensitivity Specificity

99.3% 99.99%0.5% SNV Control

Panel Lot User

User Average (n=6)

All Average (n=24)

User Average (n=6)

All Average (n=24)

1 95.5% 100.0%2 92.8% 100.0%3 94.4% 100.0%

2 1 94.3% 100.0%

0.1% SNV Control

1 94.2%

Sensitivity Specificity

100.0%

Sample Panel Lot User

Table 3. Assay reproducibility across Users, Instruments, and Reagent Lots for CNV and RNA Fusion Detection

Table 3. The reproducibility of CNV, RNA fusion and MET exon skipping detection sensitivity and specificity were demonstrated across three users, four instruments, and two lots of Pan-Cancer panel using contrived controls generated by titrating cfDNA or RNA extracted from cancer cell lines into cfTNA isolated from plasma.

Expected Fold Change

Average of Measured Fold Change (n=27)

Average Sensitivity

Expected Fold Change

Average of Measured Fold Change (n=16)

Average Specificity

MET 1.3 1.23 96% 1.0 0.99 100%EGFR 1.5 1.55 100% 1.0 1.01 100%MYC 1.3 1.22 100% 1.0 1.02 100%FGFR2 1.3 1.29 100% 1.0 1.00 100%FGFR1 1.3 1.29 100% 1.0 1.01 100%CCND1 1.3 1.39 100% 1.0 0.97 100%ERBB2 1.3 1.32 100% 1.0 0.99 100%

CNV Target

Sensitivity Specificity

MET Exon Skipping

Sensitivity (n=27)

Specificity (n=16)

MET-MET.M13M15 100 100

Fusion (~1%) Sensitivity (n=27)

Specificity (n=16)

EML4-ALK 100 100SLC34A2-ROS1 100 100CCDC6-RET 100 100

Figure 3. Case Study – Retrospective Research Study on NSCLC samples

Figure 3. Biomarker profiling using OncomineTM Pan-Cancer Cell-free Assay for longitude liquid biopsy samples collected from a human subject with NSCLC identified several gene amplifications, including MET, FGFR3, and CDK4. These observations were consistent with digital PCR and concordant with FISH analyses in solid tumor. In addition, a well-known TP53 mutation R248Q was consistently detected in the longitudinal samples with varying allelic frequencies during the time course.

MATERIALS AND METHODSPlasma and Sample preparation:Blood samples were collected into into BD K2 EDTA tubes and StreckCell-Free DNA BCT tubes following manufacturer’s instructions by a third-party vendor. Plasma was obtained by centrifugation at 1600 x g for 10 min at 4˚C, followed by another spin at 6,000 x g for 30 min at 4˚C to remove any residual blood cells.Plasma cfTNA samples: total nucleic acid was isolated from ~ 2-4 mL of plasma using MagMAX™ Cell-Free Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific, A36716) following manufacturer’s instructions. 0.1% and 0.5% SNV Control: AcroMetrix™ Oncology Hotspot Controls were diluted to either 0.1% or 0.5% allelic frequency using genomic DNA from cell line GM24385 as background. Both the control and background DNA were fragmented to mimic the size of cell-free DNA. CNV and Tri-fusion Controls: cfDNA or RNA from CNV or fusion positive cell lines were isolated and spiked into cfTNA extracted from healthy donor plasma. The CNV copy numbers were further verified by digital PCR.

Library preparation and sequencing:Targeted libraries were prepared using Oncomine™ Pan-Cancer Cell-free assay reagents (Thermo Fisher Scientific, A37664). Based on sample concentration, approximately 10-20 ng of cfTNA or equivalent 6000 copies of DNA molecules was used as input for each sample in library preparation.The Ion 540™ Kit-Chef was used for template preparation on Ion Chef™(Thermo Fisher Scientific, 4484177) and followed by sequencing on Ion GeneStuido S5 system (Thermo Fisher Scientific, A27214) using the Ion 540™ Chip (Thermo Fisher Scientific, A27766). Data analysis:Data analysis was performed using Torrent Suite™ Software v5.6 and Ion Reporter™ v5.6 for simultaneous SNV/Indel, CNV, and fusion variant calls .

Table 4 and Figure 2. Gene amplification detection in cancer cell line using Pan-Cancer cell free assay

Table 4 and Figure 2. All the expected gene amplifications were detected in diluted cfDNA samples extracted from cultured cell medium for a set of cancer cell lines. The measured fold change showed high concordance between digital PCR and Pan-Cancer cell free assay.

Sample Time Point (Months)

CNVFold Change

(Pan-Cancer Assay)Fold Change (Digital PCR)

FGFR3 1.3 1.8CDK4 1.2 1.7MET PASS PASSFGFR3 1.5 3.8CDK4 1.3 3.0MET 6.4 13.1FGFR3 1.5 4.1CDK4 1.3 3.2MET 7.6 19.0

194

256

265

8

14.5

15