Sizing up Cancer in Cell-Free DNA (A Series of Happy Accidents)

Sizing Up Cancer in Cell-Free DNA (a series of happy accidents)

Hunter Underhill Division of Medical Genetics Department of Pediatrics University of Utah December 15, 2016 Roadmap

Cell-Free DNA

GBM

Size Selection

ctDNA

Lung Cancer

Melanoma Roadmap

Cell-Free DNA

GBM

Current/ Future Size Directions Selection

ctDNA

Lung Cancer

Melanoma Glioblastoma Muliforme - Background

FLAIR Post-T1w FLAIR Post-T1w

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

http://library.med.utah.edu/WebPath/CNSHTML/CNSIDX.html#13 Glioblastoma Muliforme - Background

FLAIR Post-T1w FLAIR Post-T1w

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

http://library.med.utah.edu/WebPath/CNSHTML/CNSIDX.html#13 Glioblastoma Muliforme - Background

FLAIR Post-T1w FLAIR Post-T1w

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

http://library.med.utah.edu/WebPath/CNSHTML/CNSIDX.html#13 Glioblastoma Muliforme - Background

FLAIR Post-T1w FLAIR Post-T1w

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

http://library.med.utah.edu/WebPath/CNSHTML/CNSIDX.html#13 Glioblastoma Muliforme - Background

FLAIR Post-T1w FLAIR Post-T1w

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

Regardless of therapy, median survival remains <15 months after the initial diagnosis (Stupp et al., Lancet Oncol, 2009;10:459-66)

http://library.med.utah.edu/WebPath/CNSHTML/CNSIDX.html#13 GBM – Imaging Invasion

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

Underhill et al., NeuroImage, 2011;54:2052-65 GBM – Imaging Invasion

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

Underhill et al., NeuroImage, 2011;54:2052-65 GBM – Imaging Invasion

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

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

Cell Death Apoptotic Cell Necrotic Cell

Cell-free DNA trivia: 1. Half-life is ~10-15 minutes 2. Primary source (~80%) is circulating cells 3. 2-20 ng/mL plasma in healthy adults

100-200 bp >1,500 bp Maternal cfDNA

Red blood cell

Fetal 1. Schwarzenbach et al., Nature Rev Clinical Oncol, 2014;11:145-56 cfDNA 2. http://www.ultrasoundcare.com.au/services/nipt.html Circulating Tumor DNA – Accident #1

Human Stem Cell-Like Lines: GBM4 and GBM8 Wakimoto et al., Cancer Research, 2009;69:3472-81 No Serum Yes Serum Circulating Tumor DNA

Xenograft Model: Rat Brain – Human GBM81

f map Pre-Contrast Post-Contrast

5 144 134 human rat 4

2 122

167 % total inserts total % 1

0 fragment length (bp) Circulating Tumor DNA

Xenograft Model: Rat Brain – Human GBM83

f map Pre-Contrast Post-Contrast Circulating Tumor DNA

Xenograft Model: Rat Brain – Human GBM83

f map Pre-Contrast Post-Contrast

6 human 134 rat 5 144

167 2 153

% total inserts total % 122 1

0 fragment length (bp) Circulating Tumor DNA

Xenograft Model: Rat Brain – Human GBM

Human ctDNA Rat cell-free DNA

GBM83 6 Control2 1.5 GBM4 5 1 GBM42 4 GBM84 1.0 GBM82 3 GBM81 Control1

% total inserts total % 2 % total inserts total % 0.5 1

0 0

fragment length (bp) fragment length (bp)

GBM44 6 human 5 rat 4

2 % total inserts total % 1

0 fragment length (bp) Circulating Tumor DNA

Xenograft Model: Rat Flank – Human HCC 20x

6 human 5 rat 4

2 % total inserts total % 1

0 fragment length (bp)

Is the shift a xenograft effect? Circulating Tumor DNA

Lo et al., Sci Transl Med 2010;61ra91 Bettegowda et al., Sci Transl Med 2014;6:224ra24 Circulating Tumor DNA – Accident #2

Human Melanoma

5 134 144 human rat 4

122 2 167

0 Circulating Tumor DNA

Human Lung Cancer – Cell-Free DNA Circulating Tumor DNA

Human Lung Cancer – Sequencing Data Circulating Tumor DNA

Human Lung Cancer – ddPCR Circulating Tumor DNA

Human Lung Cancer – Fraction Selection Circulating Tumor DNA

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

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

PAGE - gBlocks

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

CoastalGenomics – Nimbus Ranger

15 AFU

Size 300 400 [bp] Size Selection

CoastalGenomics – Nimbus Ranger

15 AFU

Size 300 400 [bp] Size Selection

Nimbus Ranger – Lung Cancer T790M (N=5) LC2 Short LC2 Library T790M:WT T790M:WT 171:2450 64:2774

6.5% 2.3%

MAF MAF Ratio

WT probe intensity probe WT WT probe intensity probe WT

Mutant probe intensity Mutant probe intensity

Short Long Fraction:Library 20 Short Long

AFU LC6 Library

10 LC2 Library

Size 200 300 [bp] Size Selection

Nimbus Ranger – Lung Cancer Exon19Del (N=3)

Ex19del:WT 6:2179

0.3%

WT probe intensity probe WT MAFRatio

Mutant probe intensity

Ex19del:WT 53:911 5.5%

Fraction:Library Short Long WT probe intensity probe WT

Mutant probe intensity 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

Reminders

5 144 134 human rat 4

2 122

167 % total inserts total % 1

0 fragment length (bp)

GBM-associated cell-free DNA is GBM-associated cell-free DNA has present in plasma from a xenograft not been previously detected in brain model of GBM humans

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

Cell-Free DNA Characteristics

)

nucleosomes

free DNA] plasma)DNA] (ng/mL free

peak fragment length ( length fragmentpeak [cell GBM control GBM control GBM control lung cancer 10 GBM

6 AFU 4 Control

Size

600

500

200 300 400 1000 [bp] GBM – Accident #3

Tumor/Normal Whole Exome Sequencing (WES)

Tumor1/Normal1 Tumor2/Normal2

Variants (novel/existing): 1470 (70.5%/29.5%) Variants (novel/existing): 1108 (61.1%/38.9%)

• PTEN p.Met198del custom-designed Taqman assay for ddPCR GBM – Accident #3

PTEN p.Met198del ddPCR

PAXgene tumor DNA Buffy coat DNA Plasma cell-free DNA M198del:WT M198del:WT M198del:WT 1210:5075 1:12310 0:12753

19.3% MAF

WT probe intensity probe WT

WT probe intensity probe WT WT probe intensity probe WT

Mutant probe intensity Mutant probe intensity Mutant probe intensity

6 AFU

Size

[bp]

300

400 200 GBM – Accident #3

PTEN p.Met198del ddPCR

Fraction A Fraction B Fraction C Fraction D M198del:WT M198del:WT M198del:WT M198del:WT 1:2477 0:2612 0:2606 0:2856

probe intensity probe

WT probe intensity probe WT intensity probe WT intensity probe WT

Mutant probe intensity Mutant probe intensity Mutant probe intensity Mutant probe intensity

Fraction E Fraction F Fraction G Fraction H M198del:WT M198del:WT M198del:WT M198del:WT

0:3355 2:2975 0:2764 0:3506

WT probe intensity probe WT

WT probe intensity probe WT WT probe intensity probe WT

Mutant probe intensity Mutant probe intensity Mutant probe intensity Mutant probe intensity GBM

Intratumor Genetic Heterogeneity

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

Custom Panel (128 genes; 128 kb) ABCB1 CDKN2A FGFR3 HRAS MET QKI TERT ABCC9 CDX4 FHL2 IDH1 MMP13 RB1 TMEM147 ABL1 CIC FIP1L1 IDH2 MROH2B RET TP53 ADAM29 COL1A2 FLT3 IL18RAP MSH6 RFX6 TPTE2 AFM CTNNB1 FOXR2 IL1R2 MTOR RPL5 TRAF7 AIFM3 CXorf22 FRMD7 JAK2 NF1 SCN9A TRIM51 AKT1 CDAF12L2 FUBP1 JAK3 NF2 SEMA3C TRIM51BP ALK DDR2 FZD7 KCNC2 NLRP5 SIGLEC8 TRIM51EP ANKRD36 DRD5 GABRA1 KDR NOTCH1 SLC26A3 TRPV6 APC DYNC1I1 GABRA6 KEL NOVA1 SMAD4 UGT2A3 ATM EDIL3 GABRB2 KIT NRAS SMG5 VHL ATRX EGFR GCSAML KLF4 ODF4 SMO WNT2 BRAF ERBB2 GNA11 KRAS PARD6B SPO11 ZNF844 CALCR ERBB4 GNAQ KRTAP20-2 PDGFRA SPTA1 ZNF99 CARD6 ERCC1 GNAS LCE4A PIK3CA STAG2 CDH1 FBXW7 GOLGA5 LRRC55 PIK3R1 STK11 CDH18 FGA GPX5 LUM PLCH2 SULT1B1 CDH9 FGFR1 H3F3AP4 LZTR1 PODNL1 SYT14 CDHR3 FGFR2 HIST1H3B MAP2K1 PTEN TCHH GBM

Sequencing Metrics (N=6)

84±11 175±8 GBM

PTEN sequencing: p.Met198del

~60 Buffy

~85 Tumor

~7,900 cfDNA 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% Slide courtesy of Sabine Hellwig, PhD Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M) Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib ) 100 33.2%

a ) 100 33.2%

m l 10

e l 10

1.3% a

l LC7P1 LC7P3 1.3%

(

p LC7P1 LC7P3

y y

(

t t

y y

1 i i

t t

1 i i

2253:4537 1:9950 y s s 2253:4537 1:9950

s s

0.11% n n

0 0.11% n n

n 0.09% 0 n 0.09%

e 33.2% e

t t

e 0.1 0.1 t t

n n

i i

T 727/mL

e e

e 0.04%

b b 0.04% r

b b r 0.01

0.01 o o

o o

r r

r r 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

T T

W W

W W 800 800 600

s 727 600 400

i s 727 40 35 400 s

p e T790M probe intensity T790M probe intensity

a i 35

35 l 40 o

c T790M probe intensity T790M probe intensity p 30

a 35

L 25

c p 30

m 20 LC7P5

LC7P9

25 r

y M 15

0 10:9339 115:8688 m 20 4 T 10

LC7P5 s 3 s

LC7P9

9 2

y 15 y 5

t 7 0.11%

e 1.3%

t 4 10:9339 115:8688 0 t T 10

s 3 s

n 2 n

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

n n 35/mL

5 0.11%

e 1.3%

t 0 t

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

r 35/mL r

o r 20

W 25 -15.6% W

p p -21.1%

-31.6%

S 15

T T -36.8%

r 20

W -15.6% W

-21.1% I 10 -31.6% -31.6% -31.6% c Progession T790M probe intensity T790M probe intensity -31.6% C

S 15 -36.8%

E 5 (non-RECIST

T R lesions)

I 10 -31.6% -31.6% -31.6% Progession T790M probe intensity T790M probe intensity 0 C 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 E 5 (non-RECIST R lesions) Days on TKI therapy 0 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 Days on TKI therapy Slide courtesy of Sabine Hellwig, PhD Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M) Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib ) 100 33.2%

a ) 100 33.2%

m l 10

e l 10

1.3% a

l LC7P1 LC7P3 1.3%

(

p LC7P1 LC7P3

y y

(

t t

y y

1 i i

t t

1 i i

2253:4537 1:9950 y s s 2253:4537 1:9950

s s

0.11% n n

0 0.11% n n

n 0.09% 0 n 0.09%

e 33.2% e

t t

e 0.1 0.1 t t

n n

i i

T 727/mL

e e

e 0.04%

b b 0.04% r

b b r 0.01

0.01 o o

o o

r r

r r 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

T T

W W

W W 800 800 600

s 727 600 400

i s 727 40 35 400 s

p e T790M probe intensity T790M probe intensity

a i 35

35 l 40 o

c T790M probe intensity T790M probe intensity p 30

a 35

L 25

c p 30

m 20 LC7P5

LC7P9

25 r

y M 15

0 10:9339 115:8688 m 20 4 T 10

LC7P5 s 3 s

LC7P9

9 2

y 15 y 5

t 7 0.11%

e 1.3%

t 4 10:9339 115:8688 0 t T 10

s 3 s

n 2 n

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

n n 35/mL

5 0.11%

e 1.3%

t 0 t

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

r 35/mL r

o r 20

W 25 -15.6% W

p p -21.1%

-31.6%

S 15

T T -36.8%

r 20

W -15.6% W

-21.1% I 10 -31.6% -31.6% -31.6% c Progession T790M probe intensity T790M probe intensity -31.6% C

S 15 -36.8%

E 5 (non-RECIST

T R lesions)

NSCLC Serial Monitoring (EGFR T790M) Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib ) 100 33.2%

a ) 100 33.2%

m l 10

e l 10

1.3% a

l LC7P1 LC7P3 1.3%

(

p LC7P1 LC7P3

y y

(

t t

y y

1 i i

t t

1 i i

2253:4537 1:9950 y s s 2253:4537 1:9950

s s

0.11% n n

0 0.11% n n

n 0.09% 0 n 0.09%

e 33.2% e

t t

e 0.1 0.1 t t

n n

i i

T 727/mL

e e

e 0.04%

b b 0.04% r

b b r 0.01

0.01 o o

o o

r r

r r 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

T T

W W

W W 800 800 600

s 727 600 400

i s 727 40 35 400 s

p e T790M probe intensity T790M probe intensity

a i 35

35 l 40 o

c T790M probe intensity T790M probe intensity p 30

a 35

L 25

c p 30

m 20 LC7P5

LC7P9

25 r

y M 15

0 10:9339 115:8688 m 20 4 T 10

LC7P5 s 3 s

LC7P9

9 2

y 15 y 5

t 7 0.11%

e 1.3%

t 4 10:9339 115:8688 0 t T 10

s 3 s

n 2 n

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

n n 35/mL

5 0.11%

e 1.3%

t 0 t

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

r 35/mL r

o r 20

W 25 -15.6% W

p p -21.1%

-31.6%

S 15

T T -36.8%

r 20

W -15.6% W

-21.1% I 10 -31.6% -31.6% -31.6% c Progession T790M probe intensity T790M probe intensity -31.6% C

S 15 -36.8%

E 5 (non-RECIST

T R lesions)

NSCLC Serial Monitoring (EGFR T790M) Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib ) 100 33.2%

a ) 100 33.2%

m l 10

e l 10

1.3% a

l LC7P1 LC7P3 1.3%

(

p LC7P1 LC7P3

y y

(

t t

y y

1 i i

t t

1 i i

2253:4537 1:9950 y s s 2253:4537 1:9950

s s

0.11% n n

0 0.11% n n

n 0.09% 0 n 0.09%

e 33.2% e

t t

e 0.1 0.1 t t

n n

i i

T 727/mL

e e

e 0.04%

b b 0.04% r

b b r 0.01

0.01 o o

o o

r r

r r 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

T T

W W

W W 800 800 600

s 727 600 400

i s 727 40 35 400 s

p e T790M probe intensity T790M probe intensity

a i 35

35 l 40 o

c T790M probe intensity T790M probe intensity p 30

a 35

L 25

c p 30

m 20 LC7P5

LC7P9

25 r

y M 15

0 10:9339 115:8688 m 20 4 T 10

LC7P5 s 3 s

LC7P9

9 2

y 15 y 5

t 7 0.11%

e 1.3%

t 4 10:9339 115:8688 0 t T 10

s 3 s

n 2 n

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

n n 35/mL

5 0.11%

e 1.3%

t 0 t

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

r 35/mL r

o r 20

W 25 -15.6% W

p p -21.1%

-31.6%

S 15

T T -36.8%

r 20

W -15.6% W

-21.1% I 10 -31.6% -31.6% -31.6% c Progession T790M probe intensity T790M probe intensity -31.6% C

S 15 -36.8%

E 5 (non-RECIST

T R lesions)

NSCLC Serial Monitoring (EGFR T790M) Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib ) 100 33.2%

a ) 100 33.2%

m l 10

e l 10

1.3% a

l LC7P1 LC7P3 1.3%

(

p LC7P1 LC7P3

y y

(

t t

y y

1 i i

t t

1 i i

2253:4537 1:9950 y s s 2253:4537 1:9950

s s

0.11% n n

0 0.11% n n

n 0.09% 0 n 0.09%

e 33.2% e

t t

e 0.1 0.1 t t

n n

i i

T 727/mL

e e

e 0.04%

b b 0.04% r

b b r 0.01

0.01 o o

o o

r r

r r 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

T T

W W

W W 800 800 600

s 727 600 400

i s 727 40 35 400 s

p e T790M probe intensity T790M probe intensity

a i 35

35 l 40 o

c T790M probe intensity T790M probe intensity p 30

a 35

L 25

c p 30

m 20 LC7P5

LC7P9

25 r

y M 15

0 10:9339 115:8688 m 20 4 T 10

LC7P5 s 3 s

LC7P9

9 2

y 15 y 5

t 7 0.11%

e 1.3%

t 4 10:9339 115:8688 0 t T 10

s 3 s

n 2 n

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

n n 35/mL

5 0.11%

e 1.3%

t 0 t

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

r 35/mL r

o r 20

W 25 -15.6% W

p p -21.1%

-31.6%

S 15

T T -36.8%

r 20

W -15.6% W

-21.1% I 10 -31.6% -31.6% -31.6% c Progession T790M probe intensity T790M probe intensity -31.6% C

S 15 -36.8%

E 5 (non-RECIST

T R lesions)

NSCLC Serial Monitoring (EGFR T790M) Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib ) 100 33.2%

a ) 100 33.2%

m l 10

e l 10

1.3% a

l LC7P1 LC7P3 1.3%

(

p LC7P1 LC7P3

y y

(

t t

y y

1 i i

t t

1 i i

2253:4537 1:9950 y s s 2253:4537 1:9950

s s

0.11% n n

0 0.11% n n

n 0.09% 0 n 0.09%

e 33.2% e

t t

e 0.1 0.1 t t

n n

i i

T 727/mL

e e

e 0.04%

b b 0.04% r

b b r 0.01

0.01 o o

o o

r r

r r 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

T T

W W

W W 800 800 600

s 727 600 400

i s 727 40 35 400 s

p e T790M probe intensity T790M probe intensity

a i 35

35 l 40 o

c T790M probe intensity T790M probe intensity p 30

a 35

L 25

c p 30

m 20 LC7P5

LC7P9

25 r

y M 15

0 10:9339 115:8688 m 20 4 T 10

LC7P5 s 3 s

LC7P9

9 2

y 15 y 5

t 7 0.11%

e 1.3%

t 4 10:9339 115:8688 0 t T 10

s 3 s

n 2 n

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

n n 35/mL

5 0.11%

e 1.3%

t 0 t

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

r 35/mL r

o r 20

W 25 -15.6% W

p p -21.1%

-31.6%

S 15

T T -36.8%

r 20

W -15.6% W

-21.1% I 10 -31.6% -31.6% -31.6% c Progession T790M probe intensity T790M probe intensity -31.6% C

S 15 -36.8%

E 5 (non-RECIST

T R lesions)

NSCLC Serial Monitoring (EGFR T790M) Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib ) 100 33.2%

a ) 100 33.2%

m l 10

e l 10

1.3% a

l LC7P1 LC7P3 1.3%

(

p LC7P1 LC7P3

y y

(

t t

y y

1 i i

t t

1 i i

2253:4537 1:9950 y s s 2253:4537 1:9950

s s

0.11% n n

0 0.11% n n

n 0.09% 0 n 0.09%

e 33.2% e

t t

e 0.1 0.1 t t

n n

i i

T 727/mL

e e

e 0.04%

b b 0.04% r

b b r 0.01

0.01 o o

o o

r r

r r 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

T T

W W

W W 800 800 600

s 727 600 400

i s 727 40 35 400 s

p e T790M probe intensity T790M probe intensity

a i 35

35 l 40 o

c T790M probe intensity T790M probe intensity p 30

a 35

L 25

c p 30

m 20 LC7P5

LC7P9

25 r

y M 15

0 10:9339 115:8688 m 20 4 T 10

LC7P5 s 3 s

LC7P9

9 2

y 15 y 5

t 7 0.11%

e 1.3%

t 4 10:9339 115:8688 0 t T 10

s 3 s

n 2 n

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

n n 35/mL

5 0.11%

e 1.3%

t 0 t

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

r 35/mL r

o r 20

W 25 -15.6% W

p p -21.1%

-31.6%

S 15

T T -36.8%

r 20

W -15.6% W

-21.1% I 10 -31.6% -31.6% -31.6% c Progession T790M probe intensity T790M probe intensity -31.6% C

S 15 -36.8%

E 5 (non-RECIST

T R lesions)

NSCLC Serial Monitoring (EGFR T790M) Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib Rociletinib Rociletinib Erlotinib (375mg) (250mg) Osimeritinib ) 100 33.2%

a ) 100 33.2%

m l 10

e l 10

1.3% a

l LC7P1 LC7P3 1.3%

(

p LC7P1 LC7P3

y y

(

t t

y y

1 i i

t t

1 i i

2253:4537 1:9950 y s s 2253:4537 1:9950

s s

0.11% n n

0 0.11% n n

n 0.09% 0 n 0.09%

e 33.2% e

t t

e 0.1 0.1 t t

n n

i i

T 727/mL

e e

e 0.04%

b b 0.04% r

b b r 0.01

0.01 o o

o o

r r

r r 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 p p

T T

W W

W W 800 800 600

s 727 600 400

i s 727 40 35 400 s

p e T790M probe intensity T790M probe intensity

a i 35

35 l 40 o

c T790M probe intensity T790M probe intensity p 30

a 35

L 25

c p 30

m 20 LC7P5

LC7P9

25 r

y M 15

0 10:9339 115:8688 m 20 4 T 10

LC7P5 s 3 s

LC7P9

9 2

y 15 y 5

t 7 0.11%

e 1.3%

t 4 10:9339 115:8688 0 t T 10

s 3 s

n 2 n

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

n n 35/mL

5 0.11%

e 1.3%

t 0 t

i 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 4/mL i

r 35/mL r

o r 20

W 25 -15.6% W

p p -21.1%

-31.6%

S 15

T T -36.8%

r 20

W -15.6% W

-21.1% I 10 -31.6% -31.6% -31.6% c Progession T790M probe intensity T790M probe intensity -31.6% C

S 15 -36.8%

E 5 (non-RECIST

T R lesions)

I 10 -31.6% -31.6% -31.6% Progession T790M probe intensity T790M probe intensity 0 C 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 E 5 (non-RECIST R lesions) Days on TKI therapy 0 0 5001000 1080 1140 1200 1260 1320 1380 1440 1500 Days on TKI therapy 40 50 60 GGT A R T T GG A G C T G TTGGC G T A GGC A Y G A G T R CC

Slide courtesy of Sabine Hellwig, PhD Current/Future Directions

Pancreatic Cancer – KRAS exon 2 ice-COLD-PCR

16-0004869 Qualitative cfDNA analysis by

Sanger

NTC Healthy ctDNA NTC Healthy pool ctDNA

G T T G G C G G12V detected

Quantitative analysis by ddPCR:

G12V:WT 101:5571 1.8% 53 copies/mL

Multiplex PCR KRAS exon2 products ICP products Current/Future Directions

Genotype/Phenotype Associations

Myelin Density Imaging Dynamic MRI

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

Sabine Hellwig, PhD Randy Jensen, MD, PhD Keith Gligorich, PhD Howard Colman, MD, PhD Mary Bronner, MD

Carrie Fuertes, CRC BMP-Core Amy Hall, MB (ASCP) MLS (ASCP) John O’Shea, PhD Katy Phillips David Nix, PhD Kevin Lee Brett Milash, MS James Kline Andy Lee ARUP Brett Kennedy, PhD Funding Daniel Baker, MS • NIH K99CA168943 Elaine Gee, PhD • Department of Pediatrics research Brendan O’Fallon, PhD support funds Ashini Bolia, PhD • Shameless begging