Epigenetics:

A Scientific Frontier of
Precision & Preventative
Medicine
John B. Kisiel, MD
May 19, 2015

2010 MFMER | slide-1

Disclosures
Exact Sciences (Madison WI)
Mayo Clinic
Minor equity investor
John B. Kisiel
Scientific advisor
Research support
Potential royalties

2010 MFMER | slide-2

Goals and Objectives

Epigenetics and human disease
Epigenetics terms and concepts
Clinical applications overview
Clinical application focus: early cancer detection

2010 MFMER | slide-3

Epigenetics and Disease

10 leading causes of death in the US

Heart disease*
Cancer*
COPD*
Stroke*
Accidents
Alzheimers disease*
Diabetes*
Nephritis(-osis) *
Influenza & pneumonia
Suicide (major depression*)
*disease with an epigenetic component
2010 MFMER | slide-4

What does the term Epigenetics mean?

Changes in gene expression without a change
in DNA sequence
Gene expression is the process by which
genetic code stored in DNA is interpreted
Modifications to DNA or supporting proteins
which allow genes to be turned-on or off
Normal part of human development
Skin cell vs. blood cell have same genes but
different appearance and function (phenotype)

Aberrancy in many diseases

2010 MFMER | slide-5

Gene Expression: The Central Dogma

Change in DNA sequence (mutation) might change the protein

Altered proteins may cause disease
"Genetic code" by Madprime -. Licensed under CC BY-SA 3.0 via Wikimedia Commons-

2010 MFMER | slide-6

Regulation of Transcription: A Closer Look

Transcription factors:
Proteins that initiate the process
Must bind to the DNA upstream of transcribed code
This promotor region must be accessible

Transcription factors

"Simple transcription elongation1" by Forluvoft - Own work. Licensed under Public

Domain via Wikimedia Commons 2010 MFMER | slide-7

Epigenetics
Regulation of gene expression without change
in DNA sequence
Block access to promotor to prevent expression
Example: DNA methylation

http://missinglink.ucsf.edu/lm/genes_and_genomes/methylation.html
2010 MFMER | slide-8

Accessible DNA
DNA in one cell is 9 feet long; must fit in small package
Folded and condensed around histones (scaffolding
proteins) to form chromatin
Uncondensed DNA is where active transcription occurs
Genes under active transcription

Less active genes

"Chromatin Structures" by Original uploader was Richard Wheeler Licensed under CC BY-SA 3.0 via Wikimedia Commons 2010 MFMER | slide-9

Functional Role of Chromatin

Regulates the >25,000 protein coding genes
Each gene is tissue-specific and time-sensitive
Example:

Cell division genes must be ON for

growing embryo

Same genes ON in adult

fuel cancer growth
"Colon cancer 2" by Emmanuelm at en.wikipedia. Licensed
under CC BY 3.0 via Wikimedia Commons

"Tubal Pregnancy with embryo" by Ed Uthman, MD

(Flickr, Wikipedia)
2010 MFMER | slide-10

Epigenetics
Regulation of gene expression without change
in DNA sequence
Open or close chromatin by modifying histones
Example: Histone acetylation
Less transcription

More transcription

http://missinglink.ucsf.edu/lm/genes_and_genomes/acetylation.html
2010 MFMER | slide-11

Epigenomic Targets in Cancer Therapy

Hamm and Costa (2015) Pharmacology and Therapeutics

DNMT, DNA methyltransferase; HDAC, Histone de-acetylase
2010 MFMER | slide-12

Epigenetic Biomarkers of Cancer

Patient symptoms
Need for
biomarker

Precursor cell

Tumor cell

Early cancer

Invasive cancer

Carcinogenesis
Screening
Early detection
Cancer diangosis
Tumor profiling
Therapeutic option
Prognosis

Application of
DNA
methylation

Delpu, et al. (2013) Int. J. Mol. Sci.

2010 MFMER | slide-13

Applied Epigenetics: Colorectal Cancer

(CRC) Screening
Remains a major killer worldwide, #2 in USA
Shift toward right colon
Now ~50% R-side in US
Olmsted county: ~60% R-side

Conventional screening tools

Reduce mortality
Underutilized
Biased towards left-side

Imperative to improve
Siegel et al. CA Cancer J Clin 2015;65:5
Gupta et al. Clin Gastroenterol Hepatol. 2005;3:150
Shapiro et al. Cancer Epidemiol Biomarkers Prev. 2012;21:895

14
2010 MFMER | slide-14

Stool DNA Testing

Biological Basis

Exfoliation
Abundant
Continuous
Cancer > normal
DNA as marker
Signature changes
Stable
Amplifiable

Muco-cellular layer
Cancer

Normal

Ahlquist et al. Hum Pathol 2000

Key Technological Advances

Preservative buffer, assay sensitivity, marker selection

DNA mutations are

too heterogeneous

Olson et al. Diagn Mol Pathol 2005;14:183

Ahlquist et al. Ann Int Med 2008;149:441
http://thebluenose.com/blog/2015/02/03/
the-cell-a-molecular-approach-downloadfree/
2010 MFMER | slide-16

Key Advances: DNA Methylation Markers

Just 4 DNA methylation

markers in tissue
samples detect:
100% colon cancers
96% advanced polyps
100% specificity

Zou et al. Clin Chem 2012; 58:375

2010 MFMER | slide-17

Multi-target Stool DNA Test

Optimized & Automated

Simple device for
collection & mailing
Preservative buffer

Targets multiple markers

Methylated BMP3 & NDRG4

Mutant KRAS
-actin (human DNA)
Hemoglobin (FIT)

Sensitive multiplex DNA assay (QuARTS)

Multi-target Stool DNA Test

Addresses all elements for effective detection
High sensitivity for CRC & greatest-risk
precancer
Sensitivity
Unaffected by tumor site
Operator independent (automated)
Noninvasive
No cathartic preparation
Compliance
No diet or medication restriction
Off-site collection
Access
Widely accessible

DeeP-C study
10,000 asymptomatic patients,
91 North American centers,
Multi-target stool DNA prior to blinded colonoscopy
N Engl J Med 2014; 370:1287-97

CRC Sensitivity by Stage

MT-sDNA 94%
FIT
70%

N 29

29

21

MT-sDNA

21

10

10

NO Difference between
left and right sided CRC

60

60

CC - 21

Advanced Adenoma Sensitivity by Size

MT-sDNA

Highest risk to
progress

66

66

577

574

79

79

38

38

DNA Methylation Beyond the Colon

Highest mortality for GI cancers
50,000 CRC
100,000 foregut cancers combined

Majority are de novo

No population screening upper GI cancers
Symptoms = advanced stage disease
Novel applications for powerful marker class?

2010 MFMER | slide-23

Pancreas Cancer: A Critical Need

Pancreatic cancer (PanC): #2 cancer killer by 2020
Hypothesis: DNA sequencing would identify novel
and accurate candidate methylation markers
Aim: Test marker discrimination of PanC from
Benign pancreatic tissues
Other gastrointestinal epithelia

2010 MFMER | slide-24

Preliminary Data in Pancreas Cancer:

Discovery, technical validation
Discriminant detection (Pancreas cancer vs normals)
>500 markers met selection criteria (top 38 shown)
Many not previously reported in cancer biology

Kisiel et al. DDW 2013

2010 MFMER | slide-25

Clinical Pilot in Pancreatic Juice Aspirates

Raimondo et al. DDW 2013

2010 MFMER | slide-26

Extended Implications of Sequencing Data

Many strong DNA methylation markers without
known cancer role
Not reported in colon cancer
Not reported in pancreatic cancer
Not reported in other upper GI tract cancers

Could new markers predict cancer type?

2010 MFMER | slide-27

Tumor Site Prediction by DNA Methylation

Training set

Validation set
Kisiel et al. DDW 2013
2010 MFMER | slide-28

Summary of Site Prediction Accuracy

Predicted by Model
UGIC
LGI*
Normal
UGI Cancer (66)
Pancreas Cancer (38)

35
13
10

Colon Cancer (20)

Colon Adenoma (18)

19
14

Pancreas Normal (15)

Colon Normal (20)

Buffy Coat Normal (10)

15
20
10

Esophagus Cancer (18)

Stomach Cancer (10)

LGI* (38)
2

Controls (45)

* LGI = CRC + Adenoma > 1 cm

}
}
}

88%

(58/66)

87%

(33/38)

100%

(45/45)

2 p < 0.0001
Kisiel et al. DDW 2013
2010 MFMER | slide-29

Extended Implications of Sequencing Data

Many strong DNA methylation markers without
known cancer role
Not reported in colon cancer
Not reported in pancreatic cancer
Not reported in other upper GI tract cancers

Could new markers predict upper vs lower GI

cancers?
Could sequencing other cancers give us
anatomic location of primary cancer?

2010 MFMER | slide-30

Cornerstone Project
Anatomic Site

Lesion Category

RRBS

Validation
by qMSP

Oropharynx, lung

Tonsil, tongue
Tracheal, bronchial

In progress

Partial

Esophagus

Normal
Barretts (+/- dysplasia
Adenocarcinoma, Squamous carcinoma

Complete

Complete

Complete

Stomach

Normal
Adenoma
Adenocarcinoma

Complete

Complete

In progress

Pancreas

Normal colon, WBC, Benign pancreas

Ductal adenocarcinoma

Complete

Complete

Complete

Pancreas precursors

Main, branch duct IPMN

PanIN I-III

Complete

In progress

Bile duct

Normal bile duct, liver

Intra-hepatic CCA, Peri-hilar CCA

Complete

iCCA complete
eCCA in-progress

Liver

Hepatoma
Cirrhotic, NASH controls

In progress

Small bowel

Normal
Adenocarcinoma

In progress

Colon

Normal
SSA > 1cm, Adenoma > 1 cm, CRC

Complete

IBD

Colitis
LGD, HGD, CRC

In progress

Stool

Normal stools

Complete

Peripheral blood

Buffy coat, plasma, cellular compartments

In progress

Complete

Clinical
pilot

In progress

Complete

2010 MFMER | slide-31

Site-specificity: Expanding the scope

Best markers from each site (95)
Simultaneous multi-organ biological validation
Controls (119 tissue samples)
Neoplasia (266 tissue samples)

Analysis

Cancer vs Normal
Upper vs CRC
Pancreatico-biliary vs gastroesophageal
Combined model of entire GI tract

2010 MFMER | slide-32

Results: High Accuracy for GI Neoplasm

Chr12.133

Predicted by model
Neoplasia Control

BMP3
Any Neo
230/5

Any Neo
10/4

Neoplasia 253

13

0.049

Control

111

0.067

Accuracy

Any Neo Chr11.123

15/1

Error

0.945

CTRL
11/109

2010 MFMER | slide-33

Results: Upper GI vs Colorectal Neoplasm

Chr7.258

Predicted by model
Colorectal

Upper

Error

Colorectal 89

0.082

Upper

167

0.012

QKI
Upper
133/4
Upper
30/5

Colorectal
6/88

Accuracy

0.944

2010 MFMER | slide-34

Pancreaticobiliary vs Gastroesophageal Cancer

PDGFD

Predicted by model
Pancreatico- Gastrobiliary
esophageal

Error

Pancreaticobiliary

74

10

0.049

Gastroesophageal

78

0.067

ELOVL2
Pancreatico
-biliary

70/4
Gastroesophageal

1/70

Pancreatico
-biliary

8/0

PCPB3

Accuracy

0.941

Gastroesophageal

5/11

2010 MFMER | slide-35

Overall Model
Predicted by model
Tissue
source

CTRLs

Colorectal
neoplasia

Pancreatico- Gastrobiliary cancer esophageal

neoplasia

CTRLs

116

10

Colorectal
neoplasia

86

Pancreatico- 1
biliary cancer

65

Gastroesophageal
neoplasia

72

Overall accuracy

0.88, P<0.0001, chi-square

Kisiel, et al. AACR 2015

2010 MFMER | slide-36

Non-invasive Test: Proof of Concept

2 methylated markers (BMP3, QKI)
Non-optimized assays
2 mL of blood plasma
Predicted by model
Tissue source

Controls

Colorectal neoplasia

Pancreatic cancer

Error

Controls (n=14)

12

0.14

Colorectal neoplasia (n=14)

0.43

Pancreatic cancer (n=14)

11

0.21

Overall accuracy

0.74 (p<0.0001)

2010 MFMER | slide-37

Implications for the Future

Stop Single Organ Screening
Screen multiple cancers in a single test
Aggregate incidence outweighs organ incidence
The PERSON as the new unit of analysis
Stool test: cancers of the GI tract, lung, ENT
Blood test: all cancers

2010 MFMER | slide-38

Conclusions
Epigenetics a rich field in human disease
biology
Important implications for prevention, tumor
diagnosis, and therapy
The future is NOW: stool DNA for CRC
screening
Novel diagnostic applications:
Distinguish cancer from normal tissue
Accurately classify GI tumors by location

2010 MFMER | slide-39

Acknowledgements
Jack and Maxine Zarrow Family Foundation of Tulsa Oklahoma
Paul Calabresi Program in Clinical-Translational Research (NCI
CA90628).
Carol M. Gatton endowment for Digestive Diseases Research.
Mayo Clinic SPORE in Pancreatic Cancer (P50 CA102701),
Lustgarten Foundation for Pancreatic Cancer Research
Clinical Core of the Mayo Clinic Center for Cell Signalling in
Gastroenterology (P30DK084567).
Reagents and RRBS sequencing costs were provided by Exact
Sciences (Madison WI)

2010 MFMER | slide-40

GI Cancer Molecular Diagnostics Lab

2010 MFMER | slide-41