Sizing Up Cancer in Cell-Free DNA - University of Utah · Sizing Up Cancer in Cell-Free DNA Hunter Underhill Division of Medical Genetics Department of Pediatrics University of Utah

Sizing Up Cancer in Cell-Free DNA

Hunter UnderhillDivision of Medical Genetics

Department of PediatricsUniversity of Utah

December 15, 2016

(a series of happy accidents)

Roadmap

Cell-Free DNA

GBM

ctDNA

Melanoma

Size Selection

Lung

Cancer

Roadmap

Cell-Free DNA

GBM

ctDNA

Melanoma

Size Selection

Lung

Cancer

Current/Future

Directions

Tejada et al., J Neurooncol, 2013;116:169-175

FLAIR FLAIRPost-T1w

Dia

gno

sis

Firs

t re

curr

ence

Post-T1w

Glioblastoma Muliforme - Background

http://library.med.utah.edu/WebPath/CNSHTML/CNSIDX.html#13


FLAIR FLAIRPost-T1w

Dia

gno

sis

Firs

t re

curr

ence

Post-T1w




FLAIR FLAIRPost-T1w

Dia

gno

sis

Firs

t re

curr

ence

Post-T1w




FLAIR FLAIRPost-T1w

Dia

gno

sis

Firs

t re

curr

ence

Post-T1w




FLAIR FLAIRPost-T1w

Dia

gno

sis

Firs

t re

curr

ence

Post-T1w



Regardless of therapy, median survival remains <15 months

after the initial diagnosis (Stupp et al., Lancet Oncol, 2009;10:459-66)

Underhill et al., NeuroImage, 2011;54:2052-65

GBM – Imaging Invasion

Fast Bound-Pool Fraction Imaging (FBFI) vs. Histology







Cell-Free DNA

1. Schwarzenbach et al., Nature Rev Clinical Oncol, 2014;11:145-56

2. http://www.ultrasoundcare.com.au/services/nipt.html

Maternal cfDNA

Red blood cell

Fetal cfDNA

100-200 bp >1,500 bp

Cell Death

Apoptotic Cell Necrotic Cell

Cell-free DNA trivia:1. Half-life is ~10-15 minutes2. Primary source (~80%) is

circulating cells3. 2-20 ng/mL plasma in healthy

adults

Circulating Tumor DNA – Accident #1

Human Stem Cell-Like Lines: GBM4 and GBM8

No Serum Yes Serum

Wakimoto et al., Cancer Research, 2009;69:3472-81

Circulating Tumor DNA

Xenograft Model: Rat Brain – Human GBM81

134 144

167122

fragment length (bp)

humanrat

0

1

% to

tal in

se

rts

2

3

4

5

f map Pre-Contrast Post-Contrast







134

144

167122


humanrat

0

1

% tota

l in

sert

s

2

3

4

6

153

5


Xenograft Model: Rat Brain – Human GBM

GBM83

Control2GBM41

GBM42

GBM84

GBM82

GBM81

Control1

Human ctDNA Rat cell-free DNA

0

0.5

1.0

1.5

% to

tal in

se

rts

0

1

% to

tal in

se

rts

2

3

4

5

6

fragment length (bp) fragment length (bp)


0

1

% to

tal in

se

rts

2

3

4

5

6

humanrat

GBM44


Xenograft Model: Rat Flank – Human HCC

20x


0

1

% to

tal in

se

rts

2

3

4

5

6

humanrat

Is the shift a xenograft effect?


Bettegowda et al., Sci Transl Med 2014;6:224ra24Lo et al., Sci Transl Med 2010;61ra91

Circulating Tumor DNA – Accident #2

Human Melanoma

134 144

167122

humanrat

0

1

2

3

4

5


Human Lung Cancer – Cell-Free DNA


Human Lung Cancer – Sequencing Data


Human Lung Cancer – ddPCR


Human Lung Cancer – Fraction Selection


PLOS Genetics, 2016; 18:e1006162

Key points:

1. Cell-free DNA derived from tumor cells has a shorter fragment

length distribution in plasma compared to healthy cell-free DNA

2. Sub-fraction selection of smaller cell-free DNA fragments appears

to enrich for circulating tumor DNA

Size Selection

PAGE - gBlocks

V600E gBlocks (165 bp)T790M gBlocks (130 bp)

Size Selection

PAGE - gBlocks

V600E gBlocks (165 bp)T790M gBlocks (130 bp)

Size Selection

40

0

30

0

5

10

15

20

AF

U

Size

[bp]

CoastalGenomics – Nimbus Ranger

Size Selection

40

0

30

0

5

10

15

20

AF

U

Size

[bp]

CoastalGenomics – Nimbus Ranger

Size Selection

Nimbus Ranger – Lung Cancer T790M (N=5)

20

0

30

0

5

10

15

20

AF

U

Size

[bp]

LC2 Library

LC6 Library

LongShort

Mutant probe intensity

WT

pro

be

in

ten

sity

T790M:WT

171:2450

6.5%


WT

pro

be

in

ten

sity

T790M:WT

64:2774

2.3%

LC2 Short LC2 Library

Short Long

Fra

ction:L

ibra

ryM

AF

Ratio

Size Selection

Nimbus Ranger – Lung Cancer Exon19Del (N=3)

Short Long

Fra

ction:L

ibra

ryM

AF

Ratio

Ex19del:WT

6:2179

0.3%


WT

pro

be

in

ten

sity

Ex19del:WT

53:911

5.5%


WT

pro

be

in

ten

sity

Size Selection

Summary

• PAGE affords selection of multiple adjacent fractions with high

resolution, but is SLOW!

• Nimbus Ranger provides rapid (<6 hours) collection of 2 non-adjacent

fractions with good recovery in up to 96 samples

• Selection of shorter cell-free DNA fragments may enrich for circulating

tumor DNA in some samples, while not negatively impacting MAF in

other samples

GBM

134 144

167122


humanrat

0

1

% to

tal in

se

rts

2

3

4

5

Bettegowda et al., Sci Transl Med 2014;6:224ra24

GBM-associated cell-free DNA is present in plasma from a xenograft brain model of GBM

GBM-associated cell-free DNA has not been previously detected in humans

Reminders

GBM

Cell-Free DNA Characteristics

[ce

ll-f

ree

DN

A]

(ng

/mL

pla

sm

a)

pe

ak

fra

gm

en

t le

ng

th (

bp

)

GBM control GBM control GBM control lung

cancer

nu

cle

os

om

es

20

0

30

0

2

4

6

8

AF

U

Size

[bp]

Control

GBM10

40

0

50

0

60

0

10

00

GBM – Accident #3

Tumor/Normal Whole Exome Sequencing (WES)

Variants (novel/existing): 1470 (70.5%/29.5%)

Tumor1/Normal1 Tumor2/Normal2

Variants (novel/existing): 1108 (61.1%/38.9%)

• PTEN p.Met198del custom-designed Taqman assay for ddPCR

GBM – Accident #3

M198del:WT

1210:5075

19.3% MAF


WT

pro

be

in

ten

sity

PAXgene tumor DNA


WT

pro

be

in

ten

sity

Buffy coat DNA Plasma cell-free DNA

M198del:WT

1:12310


WT

pro

be

in

ten

sity

M198del:WT

0:12753

PTEN p.Met198del ddPCR

40

0

30

0

Size

[bp]

20

0

2

4

8

10

AF

U

6

GBM – Accident #3

PTEN p.Met198del ddPCR


WT

pro

be

in

ten

sity

Fraction A

M198del:WT

1:2477

<LOD


WT

pro

be

in

ten

sity


WT

pro

be

in

ten

sity


WT

pro

be

in

ten

sity


wT

pro

be

in

ten

sity


WT

pro

be

in

ten

sity

Fraction B

M198del:WT

0:2612

Fraction C

M198del:WT

0:2606

Fraction D

Fraction E Fraction F Fraction G Fraction H


WT

pro

be

in

ten

sity


WT

pro

be

in

ten

sity

M198del:WT

0:2856

M198del:WT

0:3506M198del:WT

0:2764M198del:WT

2:2975

M198del:WT

0:3355

GBM

Intratumor Genetic Heterogeneity

Sottoriva et al., Proc Natl Acad Sci, 2013;110:4009-14

GBM

Custom Panel (128 genes; 128 kb)

ABCB1ABCC9ABL1ADAM29AFMAIFM3AKT1ALKANKRD36APCATMATRXBRAFCALCRCARD6CDH1CDH18CDH9CDHR3

CDKN2ACDX4CICCOL1A2CTNNB1CXorf22CDAF12L2DDR2DRD5DYNC1I1EDIL3EGFRERBB2ERBB4ERCC1FBXW7FGAFGFR1FGFR2

FGFR3FHL2FIP1L1FLT3FOXR2FRMD7FUBP1FZD7GABRA1GABRA6GABRB2GCSAMLGNA11GNAQGNASGOLGA5GPX5H3F3AP4HIST1H3B

HRASIDH1IDH2IL18RAPIL1R2JAK2JAK3KCNC2KDRKELKITKLF4KRASKRTAP20-2LCE4ALRRC55LUMLZTR1MAP2K1

METMMP13MROH2BMSH6MTORNF1NF2NLRP5NOTCH1NOVA1NRASODF4PARD6BPDGFRAPIK3CAPIK3R1PLCH2PODNL1PTEN

QKIRB1RETRFX6RPL5SCN9ASEMA3CSIGLEC8SLC26A3SMAD4SMG5SMOSPO11SPTA1STAG2STK11SULT1B1SYT14TCHH

TERTTMEM147TP53TPTE2TRAF7TRIM51TRIM51BPTRIM51EPTRPV6UGT2A3VHLWNT2ZNF844ZNF99

GBM

Sequencing Metrics (N=6)

84±11 175±8

GBMB

uffy

Tu

mo

rcfD

NA

PTEN sequencing: p.Met198del

~60

~85

~7,900

GBM

Potential GBM Variants in Cell-Free DNA

GBM

Summary

• Inter-tumor genetic heterogeneity requires a personalized approach for

detecting circulating tumor DNA

• Intra-tumor genetic heterogeneity coupled with the non-metastatic

nature of GBM requires an approach with high-sensitivity for detection

of variants in cell-free DNA with a frequency <1%

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession

(non-RECIST lesions)

T790M probe intensity

WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL

Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M)

Slide courtesy of Sabine Hellwig, PhD

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL




0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL




0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL




0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL




0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL




0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL




0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500

0.01

0.1

1

10

100

T7

90M

Alle

le

Fre

qu

en

cy (p

las

ma

)

Erlotinib

Rociletinib(375mg)

Rociletinib(250mg)

33.2%

0.11% 0.09%

1.3%

Osimeritinib

0.04%

0 5001000 1080 1140 1200 1260 1320 1380 1440 150005

10152025303540

400

600

800

T7

90

M c

op

ies

per

mL

pla

sm

a 727

4 3

35

2

0 5001000 1080 1140 1200 1260 1320 1380 1440 15000

5

10

15

20

25

RE

CIS

T S

co

re

Days on TKI therapy

-15.6%

-36.8%

-31.6%

-21.1%

-31.6%

-31.6%

-31.6%Progession



WT

pro

be inte

nsity LC7P1

2253:4537

33.2%

727/mL


WT

pro

be inte

nsity LC7P3

1:9950

<LoD


WT

pro

be inte

nsity LC7P5

10:9339

0.11%

4/mL


WT

pro

be inte

nsity LC7P9

115:8688

1.3%

35/mL




G12V:WT 101:5571

1.8%

53 copies/mL

G G T A R T

40

T G G A G C T G T T

50

G G C G T A G G C A

60

Y G A G T R C C

16-0004869

GGT G C

G12V

detected

T G

NT

C

Healthy c

fDN

A

ctD

NA

Multiplex PCR products

NT

C

Healthy p

ool

ctD

NA

KRAS exon2 ICP products

Qualitative analysis by

Sanger

Quantitative analysis by ddPCR:


Pancreatic Cancer – KRAS exon 2 ice-COLD-PCR



Genotype/Phenotype Associations

Underhill, Magn Reson Med, 2016; In pressUnderhill et al., J Magn Reson Imaging, 2015;42:1611-22

Myelin Density Imaging Dynamic MRI

Conclusions

• Fragment size is important in cell-free DNA

• Overcoming challenges associated with detection of cell-free DNA

derived from GBM has profound implications for the “liquid biopsy”

Sabine Hellwig, PhDKeith Gligorich, PhDMary Bronner, MD

Carrie Fuertes, CRCAmy Hall, MB (ASCP) MLS (ASCP)

David Nix, PhDBrett Milash, MS

ARUPBrett Kennedy, PhDDaniel Baker, MSElaine Gee, PhDBrendan O’Fallon, PhDAshini Bolia, PhD

Acknowledgments

BMP-CoreJohn O’Shea, PhDKaty PhillipsKevin LeeJames KlineAndy Lee

Funding• NIH K99CA168943• Department of Pediatrics research

support funds• Shameless begging

Randy Jensen, MD, PhDHoward Colman, MD, PhD

Documents

Sizing Up Cancer in Cell-Free DNA - University of Utah · Sizing Up Cancer in Cell-Free DNA Hunter Underhill Division of Medical Genetics Department of Pediatrics University of Utah