Developing Highly Sensitive High NGS Data Efficiency ctDNA Detection Assays For Breast Cancer Surveillance

1. Introduction

A growing body of data has established the importance of monitoring dynamic changes in circulating tumor DNA (ctDNA). ctDNA detection and analysis has the potential to identify early signs of therapeutic responses, allowing for timely management of treatment to achieve more effective personalized therapy. These non-invasive ctDNA NGS tests can simultaneously map multiple genomic alterations at an affordable price. Combined with improved assay accuracy and consistency, these techniques will support more clinical validation trials and benefit more cancer patients.

2. Method

The NVIGEN X™ Cancer Precision Profiling test is a next generation sequencing (NGS) based circulating tumor DNA detection assay using a hybridization capture approach with customized gene panels. Our ctDNA NGS assay was developed with the use of high performance magnetic nanobeads, which enhances assay workflow at key steps including cfDNA extraction, NGS library preparation, and target enrichment. Experiments with individual plasma samples and DNA mutant fragments spiked in plasma samples were carried out to establish assay performance such as sensitivity, specificity, consistency and data efficiency. NGS data QC metrics of the NVIGEN assay were compared with other assays in peer reviewed publications. Tests with patient samples were also performed.

3. High Yield and Consistent cfDNA Extraction

nvigen-x-highyield-consistent-cfdna-extraction-workflow
Figure 1. cfDNA Extraction workflow.

  1. Lyse plasm @60°C
  2. Binding with MagVigen™ nanobeads at room temperature & pelleting
  3. Wash 1 & re-disperse beads pelleted in 75% EtOH
  4. Wash a 2nd time with 75% EtOH
  5. Elution of high quality cfDNA for PCR, NGS or other downstream assay

nvigen-x-cfdna-extraction-result-comparison-with-qiagen
Figure 2. We have compared our assay workflow with column-based DNA capture kit. Our workflow has shown to consistently generate higher cfDNA yield (left), better qPCR signal or less Ct (middle) and higher NGS library yield (right).

Highly Consistent and Reproducible

nvigen-x-cfdna-extraction-highly-consistent-and-reproducible bar chart
Figure 3. NVIGEN MagVigenTM cfDNA extraction was demonstrated to be highly consistent with CV% <10% across multiple plasma samples of fourplex sample sets with large or small intrinsic cfDNA contents.

4. NVIGEN X™ Cancer Precision Profiling

nvigen-x-cancer-procision-profiling
Figure 4. NVIGEN X™ Cancer Precision Profiling.

5. Highly Efficient NGS Workflow

Higher NGS Library Yield

higher-yield-and-quality-ngs-library-against-qiagen
Figure 5. With our high-performance magnetic nanobeads and optimized workflow, we were able to obtain higher yield and quality NGS Libraries from cfDNA extracted with NVIGEN Kit.

Improved NGS Data Efficiency

total-library-yield-different-input-and-elution
Figure 6. NGS library cleanup was performed using MagVigen™ – easy DNA cleanup Kit. Consistent libraries were prepared with both 10 ng and 25 ng of cfDNA input material, across various plasma samples.

6. High Sensitivity and Specificity NVIGEN X™ Assay Using DNA Spike-In Test

Lib (same Seq round of 12-plex) MAF REFDEP ALTDEP plasma spiking-in In Plasma Sample MAF
Based on NGS Data Based on Plasma Sample
lib1 0 173 0 0 0
lib2 0 177 0 0 0
lib3 0 135 0 0 0
lib4 0 142 0 0 0
lib5 0 123 0 0 0
lib6 0 82 0 0 0
lib7 0 160 0 0 0
lib9 1.7% 116 2 0 0
lib11 6.1% 123 8 8 0.07%
lib12 7.6% 121 10 8 0.07%
lib13 9% 151 15 40 0.3%
lib14 4.5% 105 5 40 0.3%

Table 1. High Sensitivity and Specificity NVIGEN X™ Assay Using DNA Spike-In Test.

nvigen-x-high-sensitivity-assay-using-dna-spike-in-test
Figure 7. DNA fragments with ESR1 D538G mutations were spiked into 2mL plasma. 8 copies and 40 copies of DNA spiking in were tested, corresponding to 0.07% and 0.3% MAF based on the plasma cfDNA content. Results showed that the NVIGEN X™ Assay detected these spiking in DNA mutants. The NGS data viewed using IGV manifest the detected spiking in DNA with the same fragment starting and ending position. With total 12 samples sequenced, the assay’s sensitivity is 100%; specificity is 90% with threshold setting at 0, and 100% if threshold setting is at 2.

7. AI Enhanced Data Technology Driving Assay Accuracy

WGS Pipeline SNP Recall SNP Precision SNP F1_score INDEL Recall INDEL Precision INDEL F1_score
NVIGEN-AI 99.9436 99.9672 99.9554 99.7513 99.8116 99.7814
KCCG-GATK 99.2091 99.9807 99.5934 99.2404 99.4446 99.3424
Broad-GATK 99.9985 99.8954 99.9379 99.5404 99.1415 99.8418
Senteion 99.9673 99.8919 99.9296 99.2143 99.3213 99.2678

Table 2. Variants called from the standard NA24385 FASTQ Comparison to the NIST GIAB true set of ~3.5M variants.

ai-enhanced-data-technology-driving-assay-accuracy-bar-chart
Figure 8. NVIGEN X™ provided highly sensitive NGS data analysis pipeline when compared with IDT assay analysis results as available from the Nature Biotech paper.

8. Assay Sensitivity with Horizon Control TruQ

Using the Horizon TruQ control sample, NVIGEN X™ assays can achieve 92% sensitivity for 10ng cfDNA input with 440X unique coverage, similar to the best performing assays reported in the Nature Biotech paper for Roche and BRP which uses >2000x unique coverage.

Input Coverage Reads Sensitivity (0.5%-2.5% VAF)
5ng 380X 0.98M 75% 18/24
10ng 440X 0.97M 92% 22/24
20ng 1525X 4.02M 1oo% 24/24
30ng 2058X 5.33M 100% 24/24

Table 3. NVIGEN X™ Assay Coverage, Reads and Sensitivity vs Input Quantities on Horizon Tru-Q7 Control Sample.

nvigen-x-assay-horizon-tru-q7-control-sample-sensitivity-vs-input-quantity
Figure 9. NVIGEN X™ Assay Sensitivity vs Input Quantities on Horizon Tru-Q7 Control Sample.

brand-name-assay-horizon-tru-q7-control-sample-sensitivity-vs-input-quantity
Figure 10. Brand Names (Roche, Illumina, IDT and BRP) Assay Sensitivity vs Input Quantities as published in the 2021 Nature Biotech Paper.

9. High Standard Quality Control

high-standard-quality-control-1
Figure 11. High Standard Quality Control 1.

high-standard-quality-control-2
Figure 12. High Standard Quality Control 2.

Heatmap and bar plots showing mutation genes in the Horizon TruQ control samples were detected accordingly with NVIGEN X™ assay. In the experiments, for 20ng and 30ng input samples, 100% of control variants covered by NVIGEN X™ -32 gene panel assay were detected. For 10 ng and 5 ng samples, 22 and 18 out of 24 control variants were detected, representing 92% and 75% sensitivity, respectively. We noted that for the variants not called in the data report, 2 for the 10ng sample, and 5 for the 5ng sample, the variants were in fact observable in IGV with call numbers >3- 4. Only 1 variant in the 5ng sample at 380X unique coverage is missing in the whole sample set, representing extremely high assay sensitivity. Better data analyzing tools are in developing with NVIGEN-AI to improve data reporting matching assay quality.

10. Artifacts Analysis of NVIGEN X™ NGS Data

artifacts-analysis-of-ngs-data
Figure 13. Artifacts Analysis of NGS Data.

Sequence artifacts (%) of all library samples. NVIGEN X™ assay provides high quality and robust NGS data. Results here shows that most of the samples are with <1-2% of sequence artifact, the fraction of alt reads indicating OxoG error induced by DNA oxidation during library preparation and is a frequent source of false-positive reading in variant calling. Patient samples (green) shows good quality of data with low artifact number.

11. Base Quality Analysis of NVIGEN X™ NGS Data

base-quality-analysis
Figure 14. Base Quality Analysis.

Sum of base quality vs. each allele base quality distribution plot shows high quality NGS data with average base quality of about 40 achieved with NVIGEN X™ assay for this set of over 40 sample experiments with various sample types and DNA input quantity (5ng to 150ng). Patient samples (green) show good data quality consistent with the rest of samples.

12. Data Efficiency by Coverage Depth and Sample Type

data-efficiency-by-coverage-depth-and-sample-type
Figure 15. Data Efficiency by Coverage Depth and Sample Type.

Data efficiency defined by paired end reads need per unique coverage is not simply correlated with coverage depth or reads or sample types but rather a fundamental assay feature reflecting the complete workflow efficiency. NVIGEN X™ assay is enabled by our highly efficient nanoparticle capture workflow5 that can achieve the ultimate data efficiency. In this set of experiments, >40 samples of different types including patient and normal plasma, whole blood, genomic DNA, body fluid and control samples at varies DNA quantity from 5ng to 150ng were evaluated with about 500x to 5000x unique coverage data per sample. Patient plasma and bodyfluid samples worked robustly as well as the rest of samples with the best patient data efficiency achieved at about 3 paired reads per unique coverage.

13. High Data Efficiency with NVIGEN X™ Assay

high-data-efficiency-with-nvigen-x-assay
Figure 16. High Data Efficiency with NVIGEN X™ Assay.

In average 10X Improvement of NGS data efficiency were achieved with NVIGEN X™ Assay compared to other brand name NGS target capture-based cancer detection assays.

The data of other brands were from Nature Biotech 2021, BMC 2020, Nature Medicine 2014.

14. A Breast Cancer Patient Case Study

Gene Report Commercial Liquid Biopsy and MAF CommercialTest Pre-Treatment (05/2021) NVIGEN X™ Pre-Treatment, Concordance with the Commercial Test. 06/22/21 NVIGEN X™ Post-Treatment, Changes with Response 07/22/21
PIK3CA: E542K 8.5% 9% N.A.
PIK3CA: E453Q 8.2% 7.4% 0.6%
ESR1: D538G 4.3% 3.3% 0.7%
ESR1: Y537N 1.4% 1.6% N.A.
ESR1: L536Q 0.4% 0.6% N.A.
ERBB2: L786V 0.3% 0.3% N.A.
ESR1: Y537S 0.3% 0.5% N.A.
ESR1: E380Q 0.4% 1.1% 0.3%
ESR1: S154L 0.7% N.A.
PIK3CA: S7L 0.4% N.A.
ESR1: P365S 0.3%
ERBB2: E79Q 0.6%

Table 4. A Breast Cancer Patient Case Study.

A 58-year-old woman was diagnosed with cT4N2M1c right breast lung cancer in March 2021. ER+/PR+/HER2 IHC2+FISH-. She received letrozole and Palbociclib 5/12/2019-6/3/2021. Liquid biopsy showed ESR1 and PIK3CA mutations. The treatment was changed to fulvestrant and alpelisib on 6/27/2021 with good clinical response. Two blood samples were tested before, and one blood sample was tested one month after the treatment. As shown in the left table, for the two blood samples collected before treatment, one (052021) was tested with a commercial liquid biopsy, the other (06222021) was tested with NVIGEN X™. Both tests showed 100% concordance on all the genes reported by the commercial assay as potentially actionable and biologically relevant that are covered by NVIGEN panel. In addition, NVIGEN X™ detected additional mutations on ESR1 and PIK3CA genes potentially due to better sensitivity and will be further evaluated with more experiments. For the blood sample (072221) collected one month after the treatment and tested with NVIGEN X™, the results were consistent for therapy response with 60% less cfDNA presenting in the blood and reduced number of genes and variants of genes when compared with the pre-treatment sample. New mutations in ESR1 and ERBB2 were also detected indicating continuous cancer cells’ evolution

15. NERNST-Seq™: Nanoparticle Enhanced RNA Spatial and Temporal Sequencing

NVIGEN NERNST-Seq™ Nanoparticle Enhanced RNA Spatial and Temporal Sequencing
Figure 17. NERNST-Seq™: Nanoparticle Enhanced RNA Spatial and Temporal Sequencing. The next evolution in single cell analysis with spatial and temporal Resolution.

16. High Fidelity Detection of Gene Variants Using Horizon Multiplex cfDNA Reference Samples

number-of-variants-detected-by-genes-at-different-allele-frequency-using-horizon-multiplex-cfdna-reference-samples
Figure 18. Number of variants detected by genes at different allele frequency using horizon multiplex cfdna reference samples.

gene-variants-detection-functional-effect-percentage-using-horizon-multiplex-cfdna-reference-samples
Figure 19. Gene variants detection functional effect percentage using horizon multiplex cfdna reference samples.

gene-variants-detection-pathogenic-drug-response-percentage-using-horizon-multiplex-cfdna-reference-samples
Figure 20. Gene variants detection pathogenic drug response percentage using horizon multiplex cfdna reference samples.

17. Gene Detection in Breast Cancer Patients

number-of-variants-detected-by-genes-per-breast-cancer-related-genes
Figure 21. Number of variants detected by genes per breast cancer related genes.

gene-detection-in-breast-cancer-patients-functional-effect-percentage
Figure 22. Gene detection in breast cancer patients functional effect percentage.

gene-detection-in-breast-cancer-patients-pathogenic-drug-response-percentage
Figure 23. Gene detection in breast cancer patients pathogenic drug response percentage.

18 conclusion

We have developed a next generation sequencing (NGS) based circulating tumor DNA detection assay using a hybridization capture approach with customized gene panels. Our assay workflow includes cfDNA extraction using NVIGEN nanoparticles, NGS library preparation, hybridization capture and NGS data analysis pipeline. Compared with other brand name products, our assay has higher cfDNA and NGS library yield. Benchmarked with published assay QC metrics, our assay has shown to have great sensitivity, high specificity, uniform consistency and 10x improved NGS data efficiency. When tested with breast cancer patient samples, our assay shows 100% concordance for important actionable and biologically relevant gene mutations reported by other commercial assays and demonstrated concordance with patient therapy response from sequential samples. NVIGEN also provides singe cell temporal and spatial RNAseq assays, and nanoparticle enhanced On-Bead-ELISA immunoassays for generating multiomics data. Our technology represents a unique and powerful platform that can best monitor cancer patients for earlier signs of drug resistance, and guide for the most effective treatment course with new therapies.

19. references

  1. Deveson, I.W., Gong, B., Lai, K. et al. Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology. Nat Biotechnol 39, 1115–1128 (2021).
  2. Verma, S., Moore, M.W., Ringler, R. et al. Analytical performance evaluation of a commercial next generation sequencing liquid biopsy platform using plasma ctDNA, reference standards, and synthetic serial dilution samples derived from normal plasma. BMC Cancer 20, 945 (2020).
  3. Newman, A., Bratman, S., To, J. et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med 20, 548–554 (2014).
  4. Costello M, Pugh TJ, Fennell TJ, et al. Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation. Nucleic Acids Research. 2013 Apr;41(6):e67. DOI: 10.1093/nar/gks1443. PMID: 23303777; PMCID: PMC3616734.
  5. NVIGEN Citations

20. Acknowledgement

The authors want to thank National Cancer Institute, National Institutes of Health (contract numbers # 75N91019C00035, # 75N91020C00052) for providing the funding to support part of this work.

The data were presented at the SABCS 2021 with poster #P2-01-15:
Developing Highly Sensitive High NGS Data Efficient ctDNA Detection Assays For Breast Cancer Surveillance, Aihua Fu1 , Wenwu Cui1, Minh V. Ton1, Kevan Wang1, Weiwei Gu1 , Tianhong Li2,Heather A. Parsons3 , Minetta C. Liu4 and George W. Sledge5 1. NVIGEN Inc., San Jose, CA; 2. UCDavis Medical Center, Davis, CA; 3. Dana-Farber Cancer Institute, Boston, CA;4. The Mayo Clinic,Rochester, MN; 5. Stanford University, Stanford, CA.