NeoGeneStar LLC

May, 2019

Thor W. Nilsen

1. Cell-free DNA (cfDNA) characteristics and purification

2. cfDNA in Cancer Diagnosis and Treatment

3. cfDNA Analysis Methods

4. Tissue of Origin Determination

5. cfDNA in Xenograft Mouse/Rat Models

Circulating Cell Free DNA

Tissue Biopsy versus Liquid Biopsy

Standard Biopsy vs Liquid Biopsy

cfDNA serves as a biomarker in clinical diagnosis:

➢ cfDNA: DNA not associated with cells

• Apoptosis related, multiples of 166bp, 332bp, 498bp

• Necrotic, pyroptotic related: 1~10kb

• Microvesicles: mostly ssDNA

• Exosomal: mostly dsDNA

• Pathogenic: DNA or RNA viruses, bacteria, etc.

➢ cfDNA in normal persons: about 1-10 ng/ml

➢ Cancer patients often have elevated cfDNA

➢ ctDNA (circulating tumor DNA) can be used as a biomarker in early detection, and monitoring of the therapeutic response of cancer treatment regimens.

➢ ctDNA is typically shorter than normal cfDNA

➢ cfDNA as biomarker in other medical conditions:

such as transplant rejection, stroke, embolism, cardiac ischemia, trauma and autoimmune diseases

Circulating Cell Free DNA• cfDNA quantity: 1-10ng/ml• cfDNA sizes: ~170bp• ctDNA: shorter

Data courtesy of Jenny Xiang, Weill Cornell Medical College

Circulating Cell Free DNAAnalysis

• qPCR – quantitative PCR• ddPCR –digital droplet PCR• Targeted (Amplicon) Sequencing• WGS -Whole Genome Sequencing• BEAMing – Beads, Emulsion,

Amplification, Magnetics • DREAMing – Discrimination of Rare

EpiAlleles by Melt• CAPP-Seq – CAncer Personalized Profiling

by deep sequencing

Cell-free Tumor DNA

Minimal Residual Diseases

Early Cancer Detection

Monitoring Tumor Burden

Cancer Mutation Tracking

Circulating Cell Free Tumor DNA

(ctDNA)

in Cancer Detection and Monitoring

Sigmoid colectomy + left lateral hepatic resection + right liver metastasis left in place

Chemo-therapyRight hepatectomy

Chemo-therapy

Time (days)

-20 0 20 40 60 80 100 120 140 160 300 400 500 600

Tota

l D

NA

fra

gm

ents

in 2

ml of

pla

sm

a (

n)

0

10000

20000

30000

40000

CT -AP-: mass in sigmoid colon and multiple liver lesions (18.0 cm)

CT -AP-: NED

Total DNA (PIK3CA)

CT -AP-: stable metastaticdisease in the right lobe of liver

CT -CAP-: 2 stable liver metastasis and 1 newlesion (10.3 cm)

CT -AP-: 2 hepatic lesions (5.8 cm)

Day 242: CT -CAP-:

NED

CT -AP-: 1 liver lesion (0.2 cm)

CT -AP-: 1 liver lesion (0.3 cm)

CT -CAP-: 1 liver lesion (0.3 cm) + 1 LULnodule (0.4 cm)

Total DNA (TP53)

SURGERY SURGERY

Data cited from Dr. Luis Diaz Johns Hopkins

Correlation of cfDNA with surgeries and cancer progression

Purification of Cell-free DNA for

Cancer Early Detection & Monitoring:

➢ Liquid Biopsy: Non-invasive - When the tumor is not accessible: cell-free DNA is the best analyte available

➢ Especially useful following surgery and/or chemotherapy:

➢ Recurrence can be detected using cell-free tumor DNA

➢ No detection of cancer signature at 6 months & 1 year is great news for patient

Diehl et al, Nature Medicine, 2008

No Detectable Cancer DNA = 100% Survival

Detection of ctDNA is predictor of poor outcome

Clinical Cancer ResearchVol. 21, Issue 14, pp 3196-3203 July 2015

NSCLC Progression Free Survival and Overall SurvivalVia Detection of cfDNA mutations

Intra-tumor Heterogeneity: How to Solve it?Circulating Cell Free Tumor DNA

Muhammed Murtaza et al Nature volume497, pages108–112 (02 May 2013)

Cell-Free Plasma DNA-Guided Treatment With Osimertinib in Patients With Advanced EGFR-Mutated NSCLCAnna Buder, et al

Diehl et al Nature Medicine, 2008

Circulating Tumor DNA Analysis

Genome equivalents (gEqs) is critical

Nanograms of cfDNA Genome Equivalents

0.33 100

1.0 300

3.3 1,000

10.0 3,000

33.0 10,000

66.0 20,000

132 40,000

•Size Selection – note cfDNA is already a perfect size•Linker adapter ligation – for amplification and tagging•Sequencing•Alignment with genome, mutational burden, microsatellite instability, etc.

Circulating Cell–free Tumor DNA Analysis

CAPP-Seq

Circulating Tumor DNA Analysis

Annals of Oncology, Volume 29, Issue 6, June 2018, Pages 1351–1353, https://doi.org/10.1093/annonc/mdy134

The content of this slide may be subject to copyright: please see the slide notes for details

Tissue of Origin Determination

• Comprehensive Human Cell TypeMethylation Atlas (Nov 2018)Nat Commun, 2018; 9: 5068.

Published online 2018 Nov 29. doi: 10.1038/s41467-018-07466-6

• Nucleosome End SequencingCell Volume 164, Issues 1–2, 14 January 2016, Pages 57-68

Volume 164, Issues 1–2, 14 January 2016, Pages 57-68

Sources of cfDNA – Tissue of Origin

Comprehensive Human Cell Type Methylation Atlas

DNA Methylation Status during Disease Status

DNA Methylation Status in Cancer Patients

DNA Methylation Status - Islet Cell Transplant

I. In Healthy Patients➢ cfDNA increases with age➢ Most (~95%) cfDNA comes from blood

cellsII. In disease states - cfDNA levels increases

➢ Cancer➢ Organ transplantation, injury➢ Stroke, sepsis, immune reaction➢ Diabetes

Methylome cfDNA Summary

➢ Cancer treatment response measurementTotal cfDNACancer specific - ctDNA

➢ Cancer of Unknown Primary Determination

cfDNA in Xenograft Mice Models

1. Circulating tumor DNA measurement provides reliable mutation detection in mice with human lung cancer xenograftsLaboratory Investigation volume 98, pages935–946 (2018)

2. MP29-05 ANDROGEN RECEPTOR GENE ABERRATIONS IN CIRCULATING CELL FREE DNA FROM JAPANESE CASTRATION RESISTANT PROSTATE CANCER PATIENTSThe Journal of UrologyVolume 199, Issue 4, Supplement, April 2018, Page e367

3. Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastomaOncotarget. 2017 Jul 7;8(49):85234-85251. doi: 10.18632/oncotarget.19076. eCollection 2017 Oct 17.

cfDNA in Xenograft Mice Models

➢Patient-Derived Tumor Xenograft (PDTX) MiceCornell Medicine -Giorgio Inghirami, M.D.

PATh PDX Facility at Cornell University

The Jackson Laboratory

Charles River

Crown Bioscience Inc.

Mitra Biotech

Champions Oncology

Bioduro

Taconic

➢OncoRat XenograftHera BioLabs

Charles River

Creative Biolabs

Crown Bioscience

Animal Models Development

cfDNA in Xenograft Mice Models

cfDNA from healthy donor

ctDNA from cancer patients Hela Mononuclosome

➢Inject Animal

➢Cancer proliferation

➢cfDNA monitoring

➢Size

➢Methylation status

➢Determination of active genes

➢Determination of tissue of origin

CUP Cancer of Unknown Primary

Circulating tumor DNA measurement provides reliable mutation detection in mice with human lung cancer xenograftsLaboratory Investigationvolume 98, pages935–946 (2018)

Biomolecular Detection and QuantificationVolume 17, March 2019, 100087The emerging role of cell-free DNA as a molecular marker for cancer management

Molecular and Cellular EndocrinologyVolume 462, Part A, 15 February 2018, Pages 17-24Patient-derived xenografts: A platform for accelerating translational research in prostate cancer

Pancreatic Adenocarcinoma Therapeutic Targets Revealed by Tumor-Stroma Cross-Talk Analyses in Patient-Derived XenograftsNicolle et al., 2017, Cell Reports21, 2458–2470

References

1. Circulating tumor DNA measurement provides reliable mutation detection in mice with human lung cancer xenograftsLaboratory Investigationvolume 98, pages935–946 (2018)

2. Biomolecular Detection and QuantificationVolume 17, March 2019, 100087

3. The emerging role of cell-free DNA as a molecular marker for cancer managementMolecular and Cellular EndocrinologyVolume 462, Part A, 15 February 2018, Pages 17-24Patient-derived xenografts: A platform for accelerating translational research in prostate cancer

4. Pancreatic Adenocarcinoma Therapeutic Targets Revealed by Tumor-Stroma Cross-Talk Analyses in Patient-Derived XenograftsNicolle et al., 2017, Cell Reports21, 2458–2470

References

I. Nucleic acid purification

➢ Between the patient and the lab

➢ Platform independent

➢ Automatable

II. NeoGeneStar cell-free DNA purification kits

➢ Simple elegant performance

➢ Scale independent

➢ Designed for typical blood draws

➢ Innovative Magnet/Sleeve Designs

NeoGeneStar LLC

Magnetic Molecular Purification

• Predispensed stabilization reagents

• Sample is added to stabilization reagent

• No need for addition of buffer and PK

• Ambient temperature stabilization of cfDNA for > 2 weeks

• Ideal for batch samples processing

Purification of Circulating Cell Free DNA using

NeoGeneStar Cell Free DNA Purification Kit

➢ Cutting edge superparamagnetic

nanotechnology

➢ Highly effective method for

cfDNA purification

➢ Simple procedures ~45 minutes

➢ No specialized equipment

required

➢ No vacuum or centrifugation

➢ Automatable

➢ Patent pending technology

➢Magnetic bead capture: no filtration,

no clogging, no need for centrifugation

➢ Expect near 100% recovery of cell-free DNA

from plasma, serum, urine and CSF

➢Highest yield of purified cell-free DNA

➢Move the magnet for wash and elution

NeoGeneStarTM Cell-free DNA Purification Kits

Key advantages of NeoGeneStarTM products:

➢ Highly effective method for cfDNA purification – no high MW artifact on electropherograms

➢ Purifies cfDNA from ~30 bases to >10,000 bases

➢ Validated for plasma, serum, urine & CSF

➢ Kits available for sample volume from 0.1 ml to 20 ml

➢ Predispersed solid formulation - minimize sample dilution

➢ Better patient sample recovery – near 100% success (compared with up to 10% failure with other methods)

➢ cfDNA storage for 2 weeks at room temperature for sample transportation, storage and batch processing

➢ NeoGeneStar cfDNA Kit purify cfDNA as low as 30bp➢ No lot to lot variation➢ Magnetic beads stable for >2 years

➢Nanodrop – concentration too low (1-100ng/ml)

➢BioAnalzer, TapeStation or Fragment Analyzer-cfDNA size distribution

➢qPCR – recommend 62bp, 170bp and 320bp

➢DNA library preparation

High MW ContaminatesInterfere with downstream reaction

CompetitorNeoGeneStar

Courtesy of Elmo Neuberger, The Johannes Gutenberg University Mainz

NeoGenestar vs. Competitor

Mostly low MW DNA degradation (Competitor)

166bp cfDNA~170bp cfDNA (NeoGeneStar)

~170bp cfDNA (NeoGeneStar)

~170bp cfDNA

Purified cfDNA Purified Urine Total DNA (not centrifuged)

~170bp cfDNA

Genomic DNA

Centrifuge Whole Blood

Transfer Plasma

➢ Compatible with all blood Collection Tubes

➢ K3EDTA, K2EDTA, Plasma Separator (PST), Streck, BioMatrica

Lbgard, etc.

➢ Stable at room temp (4 to 37 oC) for 2 weeks

➢ Ambient Shipping

➢ Storage for batch sample processing

NeoGeneStar Stabilization Tube

➢ cfDNA storage for 2 weeks at ambient temperature for

sample transportation, storage and batch process

Amenable to automation for large numbers of samples

NeoGeneStar LLC

Automation of cfDNA Purification

Stabilized Sample - Batched Sample Processing

Transfer to cfDNA binding tube (LYS tube)

Add NGS Beads + Isopropanol

Automated Pipetting Station

NeoGeneStar – cfDNA Automation Platform

Add samples to LYS tube containing lysis / binding reagents, then add Isopropanol and NGS Beads

Samples are transferred to 24 Extra Deep Well Plate

Washed 2x with wash buffer

Wash 2x with 75% ethanol

Air dry 10 elute and transfer cfDNA

Shake 30 min at Room Temperature

Move the magnet

Move the magnet

Move the magnet

NeoGeneStarTM Innovative Handheld Magnets

Magnet / Sleeve Combination

MagPen

MagStrip 8

MagSleeve 6X MagSleeve 6 MagSleeve 24

MagStrip 12 MagSleeve 96

NeoGeneStarTM Magnetic Stands

Multiform MiniMagSlide

2x 15ml – 16x 2ml – 32x 0.2ml

Multiform MidiMagSlide

4x 50ml – 6 x 15ml – 4 x 10ml96 Deep Well Magnet

24 Deep Well (10ml) Magnet 50ml x 8 Position Magnet50ml x 6 Position Magnet