Free circulating DNA as a quantitative and qualitative ... · 5 * 40m sprint exercise (5 min break) 5 * 40m sprint exercise (5 min break) 5 * 40m sprint exercise (1 min break) 75

11/30/2017

1

Perikles Simon

Johannes Gutenberg-University Mainz

Faculty of Social Science, Media and Sport

Department of Sports Medicine, Disease Prevention and Rehabilitation

[email protected]

www.sportmedizin.uni-mainz.de

Free circulating DNA as a quantitative and qualitative marker in exercise physiology

Exercise induces a highly dynamic and variable aseptic inflammation

Modified from: Walsh et al. Exercise Immunology Reviews 2010

Metabolism linkedto

Inflamation linkedto

Innate andadaptive Immunity

IL-6

TP1

TP2

Non-Linearity

Associationswith and without

Correlations

11/30/2017

2

Input

Output

gene expression

protein profile

Black

Box

Body

Setting Athlete Key-Technologies

Transcriptomics

Proteomics

metabolite profile Metabolomics

NGS Appsgenome

Histone Modifications

DNA MethylationLiquid biopsy

cell-free DNA (cfDNA)

epi-

cfDNA

DNAse I

Real-time Physiome

164 bp

Information Technology

None- Wearable

Wearable

Information Technology

The Dawning of Big Data Viru, A. A., & Viru, M. (2001). Biochemical monitoring of sport

training. Human Kinetics.

Following Moore’s law

The dawning of Big Data: “The” technological innovation

11/30/2017

3

Beating Moore’s law

The dawning of even Bigger Data: Another technological innovation

By Aaron Krol

CfDNA a „versatile“ marker or no marker at all?

Injury

Exercise

Trauma

Down-Syndrome

Cancer

Sepsis

Chronicdiseases

Elevated cfDNA levels

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4

Exercise: an aseptic inflammation associated with cfDNA increases

Walsh et al. Exercise Immunology Reviews 2010

Metabolism linkedto

Inflamation and Innate andAdaptive Immunity

cfDNA

Beiter et al. Clinical Chemistry 2011

Lactate

cfDNA and Lactate during a 40 min aerobic endurance run at 9.6 km/h

10.90.8

0.7

0.6

2

3

10 20 30 40 +15 +30

time (min)

Lacta

te (

fold

-chnge

tore

st)

***

*

1

109876

5

4

3

2

cfD

NA

(fo

ld-c

hnge to r

est)

10 20 30 40 +15 +30

time (min)

***

*

******

**

1

Lacta

te (

mm

ol/l)

10

cfD

NA

(ng/m

l)

2.5

5

20

40

2

4

8

0.610 20 30 40 +15 +30

time (min)010 20 30 40 +15 +30

time (min)0

Haller et al. IJSPP 2016

11/30/2017

5

Limit of Detection

0,03 pg DNA 1/200 Cell-DNA-content

or 1,2 ng/ml plasma

Breitbach et al. J.of Appl.Physiol. 2014

Breitbach et al. PLoS ONE 2014

Establishing ultra-sensitiv detection procedures for cfDNA

Detection of amplicons in L1PA2 repeats (14000 copies/cell)

Detection of amplicons in LTR5 repeats (ca. 1000) copies/cell)

Helmig et al. Europ. J. Appl. Physiol. 2015

Detection of amplicons in ALU repeats

Direct PCR with native plasma

Multilocus primers for highly conserved repeat regions


Routine silica-based DNA isolation looses > 60% of the cfDNA

11/30/2017

6

Breitbach et al. J Appl Physiol 2014 Mehdorn et al. IJSPP 2016

A similar study applying cycling revealed roughly 50% reducedincreases for the same O2-consumption

Subjects:

10 male triathletes

Inclusion criteria:

Complete 10 km in less than 40 min..

Preliminary investigation:

Pulmonary function test

Body impedance analysis (BIA)

Measurement of height [m] and weight [kg]

Intervention:

60% of VO2max downhill (-3.5%)

60% of VO2max uphill (6.5%)

80% of VO2max downhill (-3.5%)

80% of VO2max uphill (6.5%)

Mean SD min max

age [years] 36,9 7,03 27 48

height [m] 1,83 0,05 1,75 1,94

weight [kg] 80,18 4,49 75,1 88,2

BMI [kg/m^2] 23,97 1,49 21,94 26,92

fatty mass [%] 13,97 3,55 8,5 18,8

VC [l] 6,58 0,77 5,45 8,19

FEV1 [l] 4,98 0,65 4,01 6,38

VO2-max [ml/min/kg] 56,54 3,40 51 62,8

IAT [km/h] 13,15 1,01 12,3 15,4

V at 4mmol/l lactate [km/h] 14,03 1,19 12,6 16,4

Tab. 1: Characteristics of triathletes (N= 10).

Uphill and downhill running at same relative VO2

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Lacta

te m

mol/l (

log)

cfD

NA

ng/m

l (log)

HR

beats

/min

RE

R (

log)

At 80% VO2max downhill running ins increasing cfDNA more than uphill running

Increases in cfDNA following five repetitive sprints over 40m

9 participants (age: 23.6 ± 1.7 years; height: 179.6 ± 7.9 cm; weight: 80.9 ± 9.9 kg) 15 minute warm-up Random assignment in group A and B

5 * 40m sprint exercise(1 min break)




75 min break 75 min break

blood sampling capillary cfDNA at baseline, after warming up, after every sprint (finger tip) venous cfDNA at baseline, after series 1 and 2 lactate at baseline, after warming up, after every sprint (ear lobe)

laboratory analysis (qPCR) statistical analysis

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Results

Folie Nr. 15Datum: 30.11.2017

venous cfDNA increased on average 2.4-fold after the short pause sprints

long breaks led to a signticantly lower 1.9-fold increase after long pauses (p<0.05)

lactate increased 3.4 fold after long pause exercise and 4.9-fold after 1 minute pauses (n.s.; p≥0.258).

venous cfDNA correlated significantly with lactate (r=0.69; p<0.05).

lactate(mmol/l)

venous cfDNA(ng/ml)

*average

cfDNA long

cfDNA short

average

lactate long

lactate short

Acute and chronic cfDNA alterations following strength training

Acute:16 strength exercises; 4-15 REPs Chronic effects (values before training)

Tug et al. PLoS ONE 2017

11/30/2017

9

50

100

150

200

HR

(beats

/min

)

pre 0 1 2 3 4 5 6_post

Heart rate

6x400m, 2‘pause

6x400m, 5‘pause

6x1000m, 2‘pause

1

1,5

2

0 1 2 3 4 5 6_post

Gold Standard: Trainings Impuls (Trimp)

Trim

p

(lo

g s

cale

)6x400m, 2‘pause at 18km/h

6x400m, 5‘pause at 18km/h

6x1000m, 2‘pause at maximal lactate steady state (MLSS)

Mean velocity: 14,4 km/h; range 12,8 – 16,5 km/h

cfDNA as a biomarker for exercise load?

EE median: 636 kcal; range: 484 – 794 kcal

EE median 310 kcal; range: 227 – 379 kcal


1

1,5

2

2,5

pre

cfDNA (90bp)

cfD

NA

ng/m

l

(log s

cale

)



6x1000m, 2‘pause at maximal lactate steady state (MLSS)

Mean velocity: 14,4 km/h; range 12,8 – 16,5 km/h

cfDNA as a marker for carbohydrate energy expenditure

EE median: 636 kcal; range: 484 – 794 kcal



0

0,5

1

p3

p20

Lactate

Lacta

te m

mol/l

(lo

g s

cale

)

0 1 2 3 4 5

6_post

pre

p3

p20

0 1 2 3 4 5

6_post 1,6

1,8

2,0

2,2

2,4

2,6

2,8

1 2

log cfDNA (ng/ml)

Lo

g C

HO

(kca

l)

r =0,87; p<0.0001

”Metabolic” DNA ?

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Improved rehydration strategy for all out cycling time trial lowers cfDNA

Dehydration 16h before cycling under extrem environmental consditions in a heat chamber (air temperature 32°C, relative humidity, RH, 46%) until participants lost ca. 4 % of body mass.

Rehydration:Citrate (CIT) vs. Placebo (PLC)

Trial conducted by Martin Mooses and Vahur Oopik

Increases in cfDNA correlated with total distance covered

during game (Spearman‘s r=0.87)

Lactate vs. cfDNA during a week at baseline and following a soccer game

Lactate cfDNA

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Rykova et al. Expert Opinion on Biological Therapy 2012

Rapid release of cfDNA during exercise: Type, Origin and Function?

Beiter et al. Sports Medicine 2014

Active vesicular secretionMicroparticles /-vesiclesExosomesApoptotic bodies

Passive releaseNecrosisDetachment from cell surfaces

NetosisCell free chromatinActive release by other immune cells

Virtosomes / Metabolic DNA?

Free mitochondrial DNA ->DAMP

OriginFunction

Par

ticu

lom

ics

?

Trophic, oncogenic,immune response

Immune responseApoptosis/Metastasis

„waste disposal“

Magnetic Bead Isolation

Size ExclusionChromatography

OptiPrep densitygradient centrifugation

Extracellular vesicle and particle isolation and characterization

Alexandra Brahmer

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12

Extracellular Vesicles are released in parallel with cfDNA during exercise

Frühbeis et al. Journal of Extracellular Vesicles 2015

Eur J Appl Physiol

1 3

after subsequent centrifugation at 1600× g, 10,000× g and

100,000× g was nearly similar, demonstrating that centrifu-

gation and filtration had no critical influence on the cfDNA

levels in supernatants. Thus, the major part of the released

nuclear DNA exists in a non-vesicle-bound form in blood

plasma. Moreover, considering the low amounts of nuclear

DNA in 10,000× g and 100,000× g pellets, the contribu-

tion of EV-associated DNA to the total nuclear cfDNA pool

is marginal in healthy individuals. These findings are not

restricted to eccentric running exercise, since concentric

bicycle exercise revealed similar results (Supplemental Fig-

ure S1).

In contrast to nuclear cfDNA, we could not determine

a significant increase in mitochondrial DNA in any frac-

tion (Fig. 2b; Table 3). The majority [mean (± SD) 48.39

(21.25) %] of mitochondrial DNA was observed in the

10,000× g pellet, which is expected to include mito-

chondrial fragments pelleting together with MVs. Trace

amounts of mitochondrial DNA were identified in the

100,000× g supernatants and pellets. We detected only

(mean ± SD) 0.12 (0.11) % of total mitochondrial DNA in

the untreated 100,000× g pellets and 0.04 (0.03) % in the

DNase-treated 100,000× g pellets. In summary, most of the

cfDNA liberated in the blood stream during physical exer-

cise is released independent of EVs into the soluble plasma

fraction.

The results also permit some conclusions concerning

pre-analytical considerations. It was recommended to per-

form a first centrifugation step at 1200–1600× g and a sec-

ond step at 16,000× g to remove cellular debris which could

influence cfDNA quantification (El Messaoudi et al. 2013).

Our results indicate that this second step is not required

Fig. 2 Kinetics of mean (a)

nuclear and (b) mitochondrial

cfDNA concentrations in five

healthy subjects before and after

an incremental treadmill exer-

cise. LOQ indicates the lower

limit for the quantification of

DNA concentrations. Error bars

indicate ± SD. SN supernatant.

Total plasma, 1600× g superna-

tant; 10,000× g SN, 10,000× g

supernatant; 100,000× g SN,

100,000× g supernatant. For fur-

ther details please see Table 3

Author's personal copy

Eur J Appl Physiol

1 3

after subsequent centrifugation at 1600× g, 10,000× g and

100,000× g was nearly similar, demonstrating that centrifu-

gation and filtration had no critical influence on the cfDNA

levels in supernatants. Thus, the major part of the released

nuclear DNA exists in a non-vesicle-bound form in blood

plasma. Moreover, considering the low amounts of nuclear

DNA in 10,000× g and 100,000× g pellets, the contribu-

tion of EV-associated DNA to the total nuclear cfDNA pool

is marginal in healthy individuals. These findings are not

restricted to eccentric running exercise, since concentric

bicycle exercise revealed similar results (Supplemental Fig-

ure S1).

In contrast to nuclear cfDNA, we could not determine

a significant increase in mitochondrial DNA in any frac-

tion (Fig. 2b; Table 3). The majority [mean (± SD) 48.39

(21.25) %] of mitochondrial DNA was observed in the

10,000× g pellet, which is expected to include mito-

chondrial fragments pelleting together with MVs. Trace

amounts of mitochondrial DNA were identified in the

100,000× g supernatants and pellets. We detected only

(mean ± SD) 0.12 (0.11) % of total mitochondrial DNA in

the untreated 100,000× g pellets and 0.04 (0.03) % in the

DNase-treated 100,000× g pellets. In summary, most of the

cfDNA liberated in the blood stream during physical exer-

cise is released independent of EVs into the soluble plasma

fraction.

The results also permit some conclusions concerning

pre-analytical considerations. It was recommended to per-

form a first centrifugation step at 1200–1600× g and a sec-

ond step at 16,000× g to remove cellular debris which could

influence cfDNA quantification (El Messaoudi et al. 2013).

Our results indicate that this second step is not required

Fig. 2 Kinetics of mean (a)

nuclear and (b) mitochondrial

cfDNA concentrations in five

healthy subjects before and after

an incremental treadmill exer-

cise. LOQ indicates the lower

limit for the quantification of

DNA concentrations. Error bars

indicate ± SD. SN supernatant.

Total plasma, 1600× g superna-

tant; 10,000× g SN, 10,000× g

supernatant; 100,000× g SN,

100,000× g supernatant. For fur-

ther details please see Table 3

Author's personal copy

cfDNA / vesicDNA / vesic_surfDNA Mitochondria / cirmtDNA / DAMP

cirmtDNA

vesicDNA

vesic_surfDNA

Independent release of cfDNA and extracellular vesicles during exercise

Helmig et al. Europ. J. Appl. Physiol. 2015

11/30/2017

13

Muscle-specific hypo-methylation pattern of the MyoD core enhancer

Methylation level of MyoD core enhancer CpG2

skeletal muscle: <10% nonmuscle tissue: >50%

Brunk et al., Dev Biol. 1996

skeletalmuscle

wholeblood cells

pre post

Ø 93% (+/- 6.9)(n=14)

Ø 90% (+/- 8.1)(n=14)

23% 91%

Beiter and Simon unpublished

pre post 90+Chimerism

Blood

Chimerismus

Bone marrow

HSCT1 41 42 47 87 -

HSCT2 54 77 54 - 100

HSCT3 32 33 32 - 73

HSCT4 43 60 51 100 -

HSCT5 53 43 36 - 100

HSCT6 90+ 96+ 88+ 100 100

HSCT7 89+ 93+ 90+ 100 100

Mittelwert 57,5 63,5 56,9

SD 23,1 25,4 23,5

59,3 (±23)

X, Y X, X

Liver

HämatopoeticStem cells

X, Y

Y-chromosomal DNA: DYZ1, 77bp Botezatu et al., Clin Chem 2000

Predominant haematopoetic origin of cfDNA during exercise

Sex-missmatched transplant model

Tug et al. Exercise Immunology Reviews; 2015

11/30/2017

14

60

70

80

90

100

He

art

ra

te

(be

ats

/min

)

+8

d

36

36,4

36,8

37,2

37,6

38

Te

mp

era

ture

(°C

)

4000

6000

8000

10000

12000

-2,5

-1,5

-0,5

0,5

1,5

0,5

1

1,5

2

2,5

2,6

2,8

3

3,2

3,4

***

**

***

***

*** *

L1PA2 qPCR

PicoGreen Kitn.s.

+5

d

+3

d

+2

d

+1

d

Po

st

Sta

rt

-d

1

+8

d

+5

d

+3

d

+2

d

+1

d

Po

st

Sta

rt

-d

1

+8

d

+5

d

+3

d

+2

d

+1

d

Po

st

Sta

rt

-d

1

Le

uko

cyte

s

(n/µ

l)

+8

d

+5

d

+3

d

+2

d

+1

d

Po

st

Sta

rt

-d

1

+8

d

+5

d

+3

d

+2

d

+1

d

Po

st

Sta

rt

-d

1

+8

d

+5

d

+3

d

+2

d

+1

d

Po

st

Sta

rt

-d

1cfD

NA

ng

/ml

(lo

g s

ca

le)

hsC

RP

ng

/ml

(lo

g s

ca

le)

cfD

NA

ng

/ml

(lo

g s

ca

le)

CfDNA kinetics following cardiovascular surgery

21 Patients were monitored peri-operatively at the University Hospital in Tübingen, Germany14 extracorporal circulation (ECC)

7 without extracorporal circulation (no ECC)

Samples provided by S. Stoppelkamp and HP Wendel

-2,5

-1,5

-0,5

0,5

1,5 ECC

No ECC

hsC

RP

ng

/ml

(lo

g s

ca

le)

+8

d

+5

d

+3

d

+2

d

+1

d

Po

st

Sta

rt

-d

1

0,5

1

1,5

2

2,5

3

cfD

NA

ng

/ml

(lo

g s

ca

le)

2,6

2,8

3

3,2

3,4

+8

d

+5

d

+3

d

+2

d

+1

d

Po

st

Sta

rt

-d

1

+8

d

+5

d

+3

d

+2

d

+1

d

Po

st

Sta

rt

-d

1

**

n.s.

cfD

NA

ng

/ml

(lo

g s

ca

le) PicoGreen Kit

L1PA2 qPCR

Establishing ultra-sensitiv detection procedures for cfDNA and ctDNA

Detection of cfDNA with a KRAS mutationAs a liquid biopsy for cancer

The ideaMonitoring tumor DNA for optimizingtreatment options and sports therapy

Neuberger et al. Gene Therapy 2017

Ehlert et al. Sci. Reports 2017

Quintruple Pre-amplification followed by pooling

Transgene detection

Oncogene detection

11/30/2017

15

vv

v

v

cfDNA concentrations over the course of surgery in colon carcinoma patients

Ehlert et al. Sci. Reports 2017

cfDNA (ng/ml)

KRAS+DNA (copies/ml)

DNAse I activityreduction (%)

11/30/2017

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cfDNA concentrations over the course of exercise of colon carcinoma patients

cfDNA (ng/ml)

% KRAS+DNA (%)

1

1,5

0

25

50

75

100

12

5

po

st3

po

st5

post2

0

post3

0

Power (W)

cfDNA

0

0,5

0

25

50

75

10

0

12

5

post3

po

st5

po

st2

0

po

st3

0

Power (W)

Lactate

cfD

NA

ng/m

l

(log s

cale

)L

acta

te m

mo

l/l

(lo

g s

ca

le)

Development of lactate and cfDNA values in patients with stable coronary heart disease

Michal et al. Trials 2015

74 coronary heart disease patients: cfDNA90bp 20.1 (23.8) ng/ml; range 5.1–183.0 ng/ml 74 healthy controls: cfDNA90bp 9.7 (4.2) ng/ml; range 1.6–23.7 ng/ml


cfDNA

Lactate

11/30/2017

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1

1,5

0

25

50

75

10

0

12

5

po

st3

po

st5

po

st2

0

po

st3

0

Power (W)

cfDNA

0

0,5

0

25

50

75

100

12

5

po

st3

post5

po

st2

0

po

st3

0

Power (W)

Lactate

cfD

NA

ng/m

l

(log s

cale

)L

acta

te m

mo

l/l

(lo

g s

ca

le)

Development of lactate and cfDNA values in patients with stable coronary heart disease

Michal et al. Trials 2015

74 coronary heart disease patients: cfDNA90bp 20.1 (23.8) ng/ml; range 5.1–183.0 ng/ml 74 healthy controls: cfDNA90bp 9.7 (4.2) ng/ml; range 1.6–23.7 ng/ml


1

1,2

1,4

1,6

1,8

pre

0

du

rin

g3

du

rin

g6

du

rin

g9

du

rin

g1

2

du

rin

g1

5

po

st3

po

st5

po

st2

0

po

st3

0

0

0,2

0,4

0,6

0,8

log

lacta

te

KQ

-Mitte

lwe

rte

pre

0

du

rin

g3

du

rin

g6

du

rin

g9

du

rin

g1

2

du

rin

g1

5

po

st3

po

st5

po

st2

0

po

st3

0

cfD

NA

ng

/ml

(log s

cale

)

No CHFCHF

Haematocrit (%)

Erythrocytes (T/l)

Thrombocytes (G/l)

Immature Neutrophiles (G/l)

Mature Neutrophiles (G/l)

Eosinophiles (G/l)

Basophiles (G/l)

Monocytes (G/l)

Lymphocytes (G/l)

0,33

0,31

0,48

0,10

0,45

0,11

0,08

0,14

0,65

0,0019*

0,0040*

<,0001*

0,3937

<,0001*

0,3370

0,4574

0,1844

<,0001*

Spearman r p-Value

0,25 0,0236*

0,21 0,0609

0,41 0,0001*

0,09 0,4404

0,40 0,0002*

0,19 0,0800

0,10 0,3726

0,16 0,1424

0,62 <,0001*

Spearman r p-Value

Correlation with Capillary cfDNA Venous cfDNA

Netosis ?

Metabolic

DNAAnker et al. (1975) Spontaneous release of DNA by human blood lymphocytes as shown in an in vitro system. Cancer Research 35(9):2375–2382

Association of cfDNA with cellular populations during exercise

11/30/2017

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Rykova et al. Expert Opinion on Biological Therapy 2012

Conclusion: We are proud to say; we can measure something

Beiter et al. Sports Medicine 2014

Active vesicular secretionExosomesMicroparticles /-vesiclesApoptotic bodies

Passive releaseNecrosisDetachment from cell surfaces

NetosisCell free chromatin

Virtosomes / Metabolic DNAFree mitochondrial DNA ->DAMP

OriginFunction

Trophic, Oncogenic,Immune response

Immune response

Metastasis!???

? Myogenic ?

( )( )

cfDNA increases withCHO Energy expenditureEccentricity of exerciseProlonged duration

• Anaerobic exercise induces cell-free DNA up to 20-fold within minutes

• Cell-free DNA as well as microvesicles but not mitochondrial DNA respond to exercise in a dose-response relationship

• Cell-free DNA is a marker for carbohydrate expenditure enabling objective determination of exercise load in elite intermittent sports

• Exercise is a valuable model to optimize conditions, pre-analyticalprocessing procedures and timing for liquid biopsies

• Liquid biopsies addressing disease specific sequences and cell- andtissue-type specific sequences / methylation pattern could be a valuable tool in personalized medicine

The take home messages

11/30/2017

19

Input

Output

gene expression

protein profile

Black

Box

Body

Setting Athlete Key-Technologies

Transcriptomics

Proteomics

metabolite profile Metabolomics

NGSgenome

Histone Modifications

DNA Methylation

Establishing cfDNA as marker for personalizedsports medicine optimizing training andintervention prevention

epi-

Radiomics

Real-time Omics for everyone

Visit our Department and turn your body into a hunting gepard!

11/30/2017

20

Thank You for Your Attention!

Dr. Elmo Neuberger

Dr. Tobias Ehlert

Dr. Susanne HelmigDipl biol. Thomas Beiter

Acknowledgement

Dr. Sarah Breitbach

Dr. Suzan Tug

Dr. Ella Lachtermann

Barlo HillenDavid Ochmann

Alexandra BrahmerNils Haller

From our team

Alumini involved

Daniel Pfirrmann

Cooperation

Dr. Martin Mooses (Tartu, Estonia) Prof. Dr. Vahur Oopik (Tartu, Estonia) PD. Dr. Eva Maria Krämer-Albers (Mainz, Germany)Prof. Ines Gockel (Leipzig; Germany)Prof. Mauro Giacca (Triest; Italy)Dr. Nägele (Optoprecision; Bremen, Germany)Prof. Richard Snyder (Florida; USA)Prof. Philippe Moullier (Nantes; France)Prof. Hauke Lang (Mainz; Germany)Prof. Hans-Peter Wendel (Tübingen; Gemrany)

Funding

Optoprecision

World Anti-Doping Agency

Röttger-FoundationKalkhoff-Rose-FoundationRhine-Main-University NetworkLift-Off Fund

Boehringer-Ingelheim Fund

Federal Institute of Sports Science

Documents

Free circulating DNA as a quantitative and qualitative ... · 5 * 40m sprint exercise (5 min break) 5 * 40m sprint exercise (5 min break) 5 * 40m sprint exercise (1 min break) 75