NVIGEN X™ – Cancer Precision Profiling

Figure 1. NVIGEN X™ Cancer Precision Profiling.

NVIGEN X™ is enabled by the state-of-the-art molecularly engineered nanoparticles, MagVigen™ and MyQuVigen™, to efficiently capture and identify molecular information from proteins, cells and nucleic acids. We are developing NVIGEN X™ into accurate, automated and FDA approved clinical instruments and reagent kits deploy-able at hundreds and thousands of hospitals, turning fatal cancers into manageable diseases.

Highly Sensitive Cancer Profiling for the Precise Curative Solution

From a single vial of whole blood sample, NVIGEN X™ applies 3 streams of sample analysis:

1. CTC Phenotypic Features
2. CTC/ctDNA Genomic Alterations
3. Protein Quantification and Functions

Confirmative studies and further investigation with tissue or other biological samples and customized assays are also available.

Figure 2. NVIGEN X™ Cancer Precision Profiling with Comprehensive Circulating Markers.

CTC

• High yield capture
• High specificity
• Simultaneous immunomagnetic CTC isolation and cell surface marker detection

Figure 3. NVIGEN MagVigen™ beads (far right column) showed much higher immunomagnetic capture yield from cell spiking in whole blood samples compared to other products on market. To represent rare cell capture and generate reliable comparison data, each capture sample contained 100 H1650 cells spiking in 1 ml whole blood. Streptavidin (SA) conjugated magnetic beads were pre-incubated with biotinylated same anti-EpCAM antibody at the same antibody quantity and used for each experiment.

Figure 4. Two different types of circulating tumor cells were isolated with MyQuVigen™ fluorescent magnetic nanoparticles from a whole blood sample. Tumor cell surface markers were stained with these fluorescent magnetic nanoparticles by binding with antibodies conjugated to nanoparticle surfaces.

ctDNA

• High yield and quality extraction
• Optimal NGS library yield
• Perfect for PCR and NGS assays

Figure 5. Highly Sensitive qPCR Detection Combining NVIGEN cfDNA Extraction and Target Capture with MagVigen™ – Streptavidin Nanobeads.

Figure 6. Clean Bioanalyzer Profiles Showing Higher Yield of cfDNA Extracted with NVIGEN Kit vs. Q-ccf Kit.

Figure 7. Higher Yield and Quality NGS Library from cfDNA Extracted with NVIGEN Kit vs. Q-ccf Kit. Library Cleanup Performed Using MagVigen™ – Easy DNA Cleanup Kit.

Figure 8. 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.

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

Figure 9. 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.

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

Figure 10. NVIGEN X™ provided highly sensitive NGS data analysis pipeline when compared with IDT assay analysis results as available from the Nature Biotech paper.

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.

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

Figure 11. NVIGEN X™ Assay Sensitivity vs Input Quantities on Horizon Tru-Q7 Control Sample.

Figure 12. 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

Figure 13. High Standard Quality Control 1.

Figure 14. 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.

Figure 15. 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.

Figure 16. 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.

Figure 17. 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.

Figure 18. 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.

A Breast Cancer Patient Case Study

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 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.

Products used

• MagVigen™ – Streptavidin DNA Capture (Cat# K61002)
• MagVigen™ – cfDNA Capture (Cat# K61003)
• MagVigen™ – EASY DNA Size Select (Cat# K61001-EASY)
• MagVigen™ – Streptavidin, protein capture (Cat# 21005P)
• MagVigen™ – Steptavidin, cell separation (Cat# 21005C)
• MagVigen™ – Anti EpCAM, human (Cat# 51001)
• MagVigen™ – Antibody – customize (Cat# 51200)