ccfdna Webinar Series: The Basics and Beyond

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1 ccfdna Webinar Series: The Basics and Beyond Part I Maxwell RSC ccfdna Plasma Kit and Maxwell RSC instrument are For Research Use Only. Not for Use in Diagnostic Procedures.

2 Introduction to Circulating, Cell-Free DNA Maxwell RSC ccfdna Plasma Kit and Maxwell RSC instrument are For Research Use Only. Not for Use in Diagnostic Procedures.

3 Nucleic Acids in Plasma mirna Exosomes Circulating, cell-free DNA (ccfdna) Circulation Research. 2012;110:

4 Micro RNA Small (~22nt) non-coding RNA that function in gene regulation There are thousands of sequences of mirna Surrounded by a large group of proteins Can exist in a stable form in plasma Found in most bodily fluids The presence and amount of certain mirna can serve as biomarkers for cancer 4

Exosomes First described in 1981 Microvesicles, 40-100nm, that are expelled by cells in a regulated process Contain protein, mrna, mirna and other noncoding RNA, and possibly DNA Have been shown to

5 Exosomes First described in 1981 Microvesicles, nm, that are expelled by cells in a regulated process Contain protein, mrna, mirna and other noncoding RNA, and possibly DNA Have been shown to transport material between cells and organs Implicated in metastasis, cellular origin, immune regulation, blood coagulation, cell migration, cell differentiation Cellular and Molecular Life Sciences 2011; 68(16):

6 Exosomes in Metastasis Release of exosomes is a regulated process Destination of exosomes can lead to spread of cancer Seminars in Cancer Biology :

7 Circulating Cell-Free DNA A.K.A.: cfdna, ctdna, cfcdna Nucleosomal in size: ~170bp o Dimers and trimers can be seen Dilute: 5-30ng per ml plasma o Increases in cancer, trauma o Increased heart rate can increase levels Rapidly turned over o Half-life of 30 minutes o Allows snapshot of current genetic make up Memorial University, Faculty of Science - Biology BIOL2060/BIOL /18_21.jpg 7

8 ccfdna Timeline 8

Origins of ccfdna Secretion Cell releases DNA into bloodstream, remains alive Necrosis Caused by factors external to the cell o Infection, toxins, trauma Cell contents are released DNA degrades

9 Origins of ccfdna Secretion Cell releases DNA into bloodstream, remains alive Necrosis Caused by factors external to the cell o Infection, toxins, trauma Cell contents are released DNA degrades into nucleosomal units Apoptosis Programmed cell death Cell fragments into apoptotic bodies, which are digested by phagocytes Very little DNA released J Clin Oncol Feb 20; 32(6):

10 Other Forms of Cell-Free DNA Cell-free DNA has been detected in most biological fluids tested: o o o o o o Urine Cerebral-spinal fluid Pancreatic juice Saliva Sweat Tears Different fluids can be enriched with mutated DNA from associated organs 10

11 Differences in ccfdna from Different Sources Cell-free DNA from different sources exhibits different characteristics. Sizes of cell-free DNA from plasma and maternal urine Plasma Urine Blue: maternal Red: fetal PLoS One. 2012;7(10):e Maxwell RSC ccfdna Plasma Kit is For Research Use Only. Not for Use in Diagnostic Procedures. 11

12 Purification and Characterization of ccfdna

13 Collection of Blood from Patients 1 tube of blood typically contains: o ~10ml blood o ~4-5ml plasma 1 to 4 ml plasma is sufficient for many applications Increasing the amount of starting material increases final yield and concentration Availability of sample can be limiting 13

Blood Collection Tubes (BCTs) EDTA, Citrate, Heparin o Contain anticoagulant to prevent clotting o Stable at 4 C for up to a week o At room temperature, white blood cell lysis begins within a day

14 Blood Collection Tubes (BCTs) EDTA, Citrate, Heparin o Contain anticoagulant to prevent clotting o Stable at 4 C for up to a week o At room temperature, white blood cell lysis begins within a day Cell-Free DNA tubes o Contain a stabilizer to prevent white blood cell lysis o Stable at room temperature for 2 weeks Serum tubes o Allow clotting of proteins o White blood cells lyse LaboratoryInfo.com 14

15 Purification of ccfdna Blood is double spun (remove blood cells) ccfdna bound to matrix Wash Elute Automated/Manual purification 15

16 Quality of DNA by electrophoresis Automated electrophoresis method that allows determination of DNA size Can detect contaminating Genomic DNA Quantitation is +/- 25% Lane Starting Material 1 MW Markers 2 Plasma 3 Serum 4 Plasma + genomic DNA 16

17 Detection of ccfdna Levels of ccfdna are too low for A 260 determination dsdna specific fluorescent dyes are sensitive enough qpcr: sensitive ddpcr: sensitive, gives absolute quantitation Next-Generation Sequencing: determine genomic differences 17

18 Levels of ccfdna Cell-free DNA levels in plasma of patients with nonsmall-cell lung cancer and inflammatory lung disease Br J Cancer Jul 28; 113(3): Maxwell RSC ccfdna Plasma Kit is For Research Use Only. Not for Use in Diagnostic Procedures. 18

Increasing ccfdna Levels Through Exercise ccfdna Yield, Pre- and Post-Exercise Increased blood flow causes increased shedding of DNA into the bloodstream Blood was

19 Increasing ccfdna Levels Through Exercise ccfdna Yield, Pre- and Post-Exercise Increased blood flow causes increased shedding of DNA into the bloodstream Blood was taken from an individual before and after 60 minutes of aerobic activity and analyzed by qpcr and BioAnalyzer Results showed a transient increase in ccfdna concentration 19

purified from plasma and serum do not show an

20 Serum vs. Plasma as Source Clotting allows lysis of WBCs, greatly increasing DNA yields with contaminating gdna BioAnalyzer Analysis In matched maternal samples, DNA purified from plasma and serum do not show an increase in fetal DNA levels DNA from Plasma and Serum, Same Donor Plasma Serum 20

21 Applications of ccfdna

pg/ul fetal DNA Both Maternal and Fetal ccfdna are Present in Pregnant Women Fetal ccfdna is present in maternal plasma Levels can increase to ~25% of total

22 pg/ul fetal DNA Both Maternal and Fetal ccfdna are Present in Pregnant Women Fetal ccfdna is present in maternal plasma Levels can increase to ~25% of total ccfdna Rapidly cleared after birth Detection of Fetal DNA in Maternal Plasma week of pregnancy sonicgenetics.com.au 22

Non-Invasive Prenatal Testing DNA is purified from the mother s plasma Fetal DNA is distinguished from maternal DNA o Paternal markers o Epigenetic markers Fetal DNA can be detected in the first

23 Non-Invasive Prenatal Testing DNA is purified from the mother s plasma Fetal DNA is distinguished from maternal DNA o Paternal markers o Epigenetic markers Fetal DNA can be detected in the first weeks of pregnancy o Same size as maternal DNA own_rump_lengh_12_weeks_ecografia_dr._wolfgang_morod er.jpg Maxwell RSC ccfdna Plasma Kit is For Research Use Only. Not for Use in Diagnostic Procedures. 23

24 Fetal Applications of ccfdna Replacing amniocentesis o Detection of trisomy o Determine gender through Y chromosome detection o Detect marker for various X-linked conditions o Detection of Rh factor SNP Analysis o Paternity o Epigenetic fetal markers Maxwell RSC ccfdna Plasma Kit is For Research Use Only. Not for Use in Diagnostic Procedures. 24

ccfdna Use in Organ Transplants Heart transplants: 25-30% rejection in first year Endomyocardial biopsy (EMB) is the current gold standard. Removes a small piece of the myocardium.

25 ccfdna Use in Organ Transplants Heart transplants: 25-30% rejection in first year Endomyocardial biopsy (EMB) is the current gold standard. Removes a small piece of the myocardium. o EMB is widely used for surveillance of cardiac allograft rejection o Recipients receive multiple rounds of EMB 25

Using ccfdna as a Rejection Marker ccfdna can be used as a marker for rejection As donor ccfdna increases, rejection risk rises ccfdna is extracted from donor, purified, and

26 Using ccfdna as a Rejection Marker ccfdna can be used as a marker for rejection As donor ccfdna increases, rejection risk rises ccfdna is extracted from donor, purified, and sequenced Levels of donor DNA are determined Sci Transl Med Jun 18; 6(241): 241ra77. Maxwell RSC ccfdna Plasma Kit is For Research Use Only. Not for Use in Diagnostic Procedures. 26

Oncological Applications of ccfdna DNA from tumor cells is released into the blood where it is degraded Cancer patients frequently exhibit elevated levels of ccfdna First described

27 Oncological Applications of ccfdna DNA from tumor cells is released into the blood where it is degraded Cancer patients frequently exhibit elevated levels of ccfdna First described in cancer patients in 1977 Recent advances in sequencing and PCR have made ccfdna a powerful oncological tool 27

28 Tumor Biomarkers in Plasma Microsatellite instability o Genetic hypermutability (predisposition to mutation) that results from impaired DNA mismatch repair (MMR) Epigenetic patterns o Changes in DNA and chromatin not related to DNA sequence. Mutations o KRAS o BRCA o SMD4 Maxwell RSC ccfdna Plasma Kit and Microsatellite Instability Analysis (MSI) are For Research Use Only. Not for Use in Diagnostic Procedures. 28

29 ccfdna and Microsatellite Instability Microsatellite: area of DNA repeats; 1-6 bp sequence repeats 5-50 times. MSI: genetic predisposition to mutation that results from impaired DNA mismatch repair (MMR). Certain MSIs are linked to certain cancers, especially colon. Representative capillary electrophoresis result for MSIHigh colorectal polyp 267B using long mononucleotides Bacher, et al. (2014). Potential new markers for detection of MSI in polyps and endometrial cancers. Maxwell RSC ccfdna Plasma Kit and Microsatellite Instability Analysis (MSI) are For Research Use Only. Not for Use in Diagnostic Procedures. 29

ccfdna and Epigenetics Epigenetics: All heritable changes in DNA/chromatin structure that are not coded in DNA itself o o o Methylation, and acetylation of both DNA and

30 ccfdna and Epigenetics Epigenetics: All heritable changes in DNA/chromatin structure that are not coded in DNA itself o o o Methylation, and acetylation of both DNA and histones, RNA-mediated silencing. Methylation occurs on cytosine bases. Hyper- and hypo-methylation, and other modifications, leads to inappropriate gene expression. 30

31 Types of Tumor Biomarkers Prognostic Objectively evaluate the patient s overall outcome, such as the probability of cancer recurrence after standard treatment Useful for the selection of patients for treatment but does not directly predict the response to a treatment Predictive Determine the potential success rate of different types of treatment. Maxwell RSC ccfdna Plasma Kit is For Research Use Only. Not for Use in Diagnostic Procedures. 31

32 Detection of Mutations Percent of a sequence of DNA that is mutated usually correlates to the stage of the cancer. At early stages, mutation levels are 0.1%, increasing to >50% in later stages. Early detection of cancer requires sensitive detection levels. Mutations indicating a specific cancer are enriched in fluids associated with that organ: Urine: CSF: Pancreatic juice: Saliva: colon, kidney, prostate cancers brain cancer pancreatic cancer esophageal, stomach, oral cancers 32

33 Mutation Detection Levels qpcr, ddpcr and Next-Generation Sequencing (NGS) Comput Struct Biotechnol J Jun 1;14:

34 Amount of DNA Needed for Mutation Detection One copy (or genomic equivalent) = 3.3pg 1000 copies = 3 4pg 34

fixation Centrifuge for plasma Tumor embedding DNA Invasive

35 The Liquid Biopsy A less invasive biopsy than standard tissue biopsy Tissue Biopsy Liquid Biopsy Excised tissue Blood fixation Centrifuge for plasma Tumor embedding DNA Invasive Expensive Slow (time consuming) Tumor Safe Less expensive Rapid DNA 35

36 Additional Advantages of a Liquid Biopsy Heterogeneity of tumor cells o Early in progression it is very possible to miss cancerous tissue Homogeneity of plasma ccfdna Ability to detect secondary cancers by biomarkers Ease of sample collection Can take repeated samples sie_lunge_computertomographie_bc.png 36

37 Disadvantages of a Liquid Biopsy Heterogeneity in the amount of circulating DNA Uncertain source/location of cells producing the ccfdna Contaminating normal DNA may obscure detection of cancer/mutated ccfdna Somatic mutations are only detected in 70-80% of nonmetastatic cancers sie_lunge_computertomographie_bc.png 37

38 Repeated Sampling by Liquid Biopsy Enables Simple Assessment Over Time Ability to take repeated blood samples throughout treatment allows for: Earlier detection More samples from more patients Detection of actionable mutations Assessment of changes in mutation levels over time 38

Levels of Breast Cancer Mutations in a treated Patient As cancer is treated, general levels of ccfdna decrease If a relapse occurs, ccfdna levels can increase Once increased levels are

39 Levels of Breast Cancer Mutations in a treated Patient As cancer is treated, general levels of ccfdna decrease If a relapse occurs, ccfdna levels can increase Once increased levels are detected, a different treatment can be pursued N Engl J Med Mar 28;368(13): Maxwell RSC ccfdna Plasma Kit is For Research Use Only. Not for Use in Diagnostic Procedures. 39

40 Liquid Biopsy and Precision Medicine Millions of tissue biopsies are performed yearly in the U.S. Liquid biopsies could provide an inexpensive way to screen for early detection of cancers As more data is generated, more biomarkers will be discovered NCI budget includes funds to monitor treatment over time using ccfdna accelerate biomedical discoveries and provide clinicians with new tools, knowledge, and therapies to select which treatments will work best for which patients. Many companies, both established and start-ups, are focusing on precision medicine and ccfdna to improve treatments of individual patients 40

cell lung cancer who do not have mutations in EGFR or ALK genes typically

41 Example of Precision Medicine Pembrolizumab (Keytruda) o humanized monoclonal antibody blocking the T-cell gene PD1 Patients with non-small cell lung cancer who do not have mutations in EGFR or ALK genes typically see positive effects when treated Efficacy of treatment is based on genetic markers 41

42 Summary ccfdna is dilute, highly fragmented, and a transient component of plasma ccfdna levels are increased by exercise, trauma, infection, or the presence of cancer Fetal ccfdna is a component of maternal plasma o Purification of ccfdna from maternal plasma allows analysis of fetal abnormalities such as trisomy Cancer biomarkers can be detected in ccfdna, allowing greatly improved treatment: o o o o Early detection Determination of cancer type Monitoring cancer levels Effectiveness of treatment Maxwell RSC ccfdna Plasma Kit is For Research Use Only. Not for Use in Diagnostic Procedures. 42

43 Coming soon ccfdna Webinar Series: The Basics and Beyond Part 2 ccfdna Workflows: Honing in on the Target Part 3 ccfdna In the Lab: Optimizing Purification for Sequencing Maxwell RSC ccfdna Plasma Kit and Maxwell RSC instrument are For Research Use Only. Not for Use in Diagnostic Procedures.

Products for cfdna and mirna isolation. Subhead Circulating Cover nucleic acids from plasma

MACHEREY-NAGEL Products for cfdna and mirna isolation Bioanalysis Subhead Circulating Cover nucleic acids from plasma n Flexible solutions for small and large blood plasma volumes n Highly efficient recovery