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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

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SLIDE 1

Sizing Up Cancer in Cell-Free DNA

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

(a series of happy accidents)

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SLIDE 2

Roadmap

Cell-Free DNA GBM ctDNA

Melanoma

Size Selection

Lung Cancer

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SLIDE 3

Roadmap

Cell-Free DNA GBM ctDNA

Melanoma

Size Selection

Lung Cancer

Current/ Future Directions

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SLIDE 4

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

FLAIR FLAIR Post-T1w Diagnosis First recurrence Post-T1w

Glioblastoma Muliforme - Background

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

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SLIDE 5

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

FLAIR FLAIR Post-T1w Diagnosis First recurrence Post-T1w

Glioblastoma Muliforme - Background

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

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SLIDE 6

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

FLAIR FLAIR Post-T1w Diagnosis First recurrence Post-T1w

Glioblastoma Muliforme - Background

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

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SLIDE 7

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

FLAIR FLAIR Post-T1w Diagnosis First recurrence Post-T1w

Glioblastoma Muliforme - Background

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

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SLIDE 8

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

FLAIR FLAIR Post-T1w Diagnosis First recurrence Post-T1w

Glioblastoma Muliforme - Background

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

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

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SLIDE 9

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

GBM – Imaging Invasion

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

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

GBM – Imaging Invasion

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

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SLIDE 11

GBM – Imaging Invasion

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

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

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SLIDE 12

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 minutes
  • 2. Primary source (~80%) is

circulating cells

  • 3. 2-20 ng/mL plasma in healthy

adults

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SLIDE 13

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

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Circulating Tumor DNA

Xenograft Model: Rat Brain – Human GBM81

134 144 167

122

fragment length (bp)

human rat

1

% total inserts

2 3 4 5

f map Pre-Contrast Post-Contrast

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SLIDE 15

Circulating Tumor DNA

Xenograft Model: Rat Brain – Human GBM83

f map Pre-Contrast Post-Contrast

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SLIDE 16

Circulating Tumor DNA

Xenograft Model: Rat Brain – Human GBM83

f map Pre-Contrast Post-Contrast

134 144 167

122

fragment length (bp)

human rat

1

% total inserts

2 3 4 6

153

5

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SLIDE 17

Circulating Tumor DNA

Xenograft Model: Rat Brain – Human GBM

GBM83 Control2 GBM41 GBM42 GBM84 GBM82 GBM81 Control1

Human ctDNA Rat cell-free DNA

0.5 1.0 1.5

% total inserts

1

% total inserts

2

3

4 5 6

fragment length (bp) fragment length (bp) fragment length (bp)

1

% total inserts

2

3

4 5 6

human rat

GBM44

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Circulating Tumor DNA

Xenograft Model: Rat Flank – Human HCC

20x

fragment length (bp)

1

% total inserts

2

3

4 5 6

human rat

Is the shift a xenograft effect?

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SLIDE 19

Circulating Tumor DNA

Bettegowda et al., Sci Transl Med 2014;6:224ra24 Lo et al., Sci Transl Med 2010;61ra91

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SLIDE 20

Circulating Tumor DNA – Accident #2

Human Melanoma

134 144 167

122 human rat

1 2 3 4 5

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SLIDE 21

Circulating Tumor DNA

Human Lung Cancer – Cell-Free DNA

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SLIDE 22

Circulating Tumor DNA

Human Lung Cancer – Sequencing Data

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SLIDE 23

Circulating Tumor DNA

Human Lung Cancer – ddPCR

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SLIDE 24

Circulating Tumor DNA

Human Lung Cancer – Fraction Selection

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SLIDE 25

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

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SLIDE 26

Size Selection

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

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SLIDE 27

Size Selection

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

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SLIDE 28

Size Selection

400 300 5 10 15 20

AFU Size [bp]

CoastalGenomics – Nimbus Ranger

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Size Selection

400 300 5 10 15 20

AFU Size [bp]

CoastalGenomics – Nimbus Ranger

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Size Selection

Nimbus Ranger – Lung Cancer T790M (N=5)

200 300 5 10 15 20

AFU Size [bp]

LC2 Library LC6 Library Long Short

Mutant probe intensity WT probe intensity T790M:WT 171:2450

6.5%

Mutant probe intensity WT probe intensity T790M:WT 64:2774

2.3% LC2 Short LC2 Library

Short Long Fraction:Library MAF Ratio

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SLIDE 31

Size Selection

Nimbus Ranger – Lung Cancer Exon19Del (N=3)

Short Long Fraction:Library MAF Ratio

Ex19del:WT 6:2179

0.3%

Mutant probe intensity WT probe intensity Ex19del:WT 53:911

5.5%

Mutant probe intensity WT probe intensity

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SLIDE 32

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

  • ther samples
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SLIDE 33

GBM

134 144 167

122

fragment length (bp)

human rat

1

% total inserts

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

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SLIDE 34

GBM

Cell-Free DNA Characteristics

[cell-free DNA] (ng/mL plasma) peak fragment length (bp)

GBM control GBM control GBM control lung cancer

nucleosomes

200 300 2 4 6 8

AFU Size [bp]

Control GBM

10 400 500 600 1000

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SLIDE 35

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
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GBM – Accident #3

M198del:WT 1210:5075 19.3% MAF Mutant probe intensity WT probe intensity

PAXgene tumor DNA

Mutant probe intensity WT probe intensity

Buffy coat DNA Plasma cell-free DNA

M198del:WT 1:12310 Mutant probe intensity WT probe intensity M198del:WT 0:12753

PTEN p.Met198del ddPCR

400 300

Size [bp]

200 2 4 8 10

AFU

6

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SLIDE 37

GBM – Accident #3

PTEN p.Met198del ddPCR

Mutant probe intensity WT probe intensity

Fraction A

M198del:WT 1:2477 <LOD Mutant probe intensity WT probe intensity Mutant probe intensity WT probe intensity Mutant probe intensity WT probe intensity Mutant probe intensity wT probe intensity Mutant probe intensity WT probe intensity

Fraction B

M198del:WT 0:2612

Fraction C

M198del:WT 0:2606

Fraction D Fraction E Fraction F Fraction G Fraction H

Mutant probe intensity WT probe intensity Mutant probe intensity WT probe intensity M198del:WT 0:2856 M198del:WT 0:3506 M198del:WT 0:2764 M198del:WT 2:2975 M198del:WT 0:3355

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SLIDE 38

GBM

Intratumor Genetic Heterogeneity

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

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SLIDE 39

GBM

Custom Panel (128 genes; 128 kb)

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

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SLIDE 40

GBM

Sequencing Metrics (N=6)

84±11 175±8

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SLIDE 41

GBM

Buffy Tumor cfDNA

PTEN sequencing: p.Met198del

~60 ~85 ~7,900

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SLIDE 42

GBM

Potential GBM Variants in Cell-Free DNA

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SLIDE 43

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

  • f variants in cell-free DNA with a frequency <1%
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SLIDE 44

T790M probe intensity WT probe intensity

LC7P1 2253:4537 33.2% 727/mL

T790M probe intensity WT probe intensity

LC7P3 1:9950 <LoD

T790M probe intensity WT probe intensity

LC7P5 10:9339 0.11% 4/mL

T790M probe intensity WT probe intensity

LC7P9 115:8688 1.3% 35/mL

5001000 1080 1140 1200 1260 1320 1380 1440 1500 0.01 0.1 1 10 100

T790M Allele Frequency (plasma)

Erlotinib Rociletinib (375mg) Rociletinib (250mg) 33.2% 0.11% 0.09% 1.3% Osimeritinib 0.04%

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25 30 35 40 400 600 800

T790M copies per mL plasma

727 4 3 35 2

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25

RECIST Score

Days on TKI therapy

  • 15.6%
  • 36.8%
  • 31.6%
  • 21.1%
  • 31.6%
  • 31.6%
  • 31.6%

Progession (non-RECIST lesions)

Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M)

Slide courtesy of Sabine Hellwig, PhD

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SLIDE 45

T790M probe intensity WT probe intensity

LC7P1 2253:4537 33.2% 727/mL

T790M probe intensity WT probe intensity

LC7P3 1:9950 <LoD

T790M probe intensity WT probe intensity

LC7P5 10:9339 0.11% 4/mL

T790M probe intensity WT probe intensity

LC7P9 115:8688 1.3% 35/mL

5001000 1080 1140 1200 1260 1320 1380 1440 1500 0.01 0.1 1 10 100

T790M Allele Frequency (plasma)

Erlotinib Rociletinib (375mg) Rociletinib (250mg) 33.2% 0.11% 0.09% 1.3% Osimeritinib 0.04%

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25 30 35 40 400 600 800

T790M copies per mL plasma

727 4 3 35 2

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25

RECIST Score

Days on TKI therapy

  • 15.6%
  • 36.8%
  • 31.6%
  • 21.1%
  • 31.6%
  • 31.6%
  • 31.6%

Progession (non-RECIST lesions)

Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M)

Slide courtesy of Sabine Hellwig, PhD

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SLIDE 46

T790M probe intensity WT probe intensity

LC7P1 2253:4537 33.2% 727/mL

T790M probe intensity WT probe intensity

LC7P3 1:9950 <LoD

T790M probe intensity WT probe intensity

LC7P5 10:9339 0.11% 4/mL

T790M probe intensity WT probe intensity

LC7P9 115:8688 1.3% 35/mL

5001000 1080 1140 1200 1260 1320 1380 1440 1500 0.01 0.1 1 10 100

T790M Allele Frequency (plasma)

Erlotinib Rociletinib (375mg) Rociletinib (250mg) 33.2% 0.11% 0.09% 1.3% Osimeritinib 0.04%

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25 30 35 40 400 600 800

T790M copies per mL plasma

727 4 3 35 2

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25

RECIST Score

Days on TKI therapy

  • 15.6%
  • 36.8%
  • 31.6%
  • 21.1%
  • 31.6%
  • 31.6%
  • 31.6%

Progession (non-RECIST lesions)

Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M)

Slide courtesy of Sabine Hellwig, PhD

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SLIDE 47

T790M probe intensity WT probe intensity

LC7P1 2253:4537 33.2% 727/mL

T790M probe intensity WT probe intensity

LC7P3 1:9950 <LoD

T790M probe intensity WT probe intensity

LC7P5 10:9339 0.11% 4/mL

T790M probe intensity WT probe intensity

LC7P9 115:8688 1.3% 35/mL

5001000 1080 1140 1200 1260 1320 1380 1440 1500 0.01 0.1 1 10 100

T790M Allele Frequency (plasma)

Erlotinib Rociletinib (375mg) Rociletinib (250mg) 33.2% 0.11% 0.09% 1.3% Osimeritinib 0.04%

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25 30 35 40 400 600 800

T790M copies per mL plasma

727 4 3 35 2

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25

RECIST Score

Days on TKI therapy

  • 15.6%
  • 36.8%
  • 31.6%
  • 21.1%
  • 31.6%
  • 31.6%
  • 31.6%

Progession (non-RECIST lesions)

Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M)

Slide courtesy of Sabine Hellwig, PhD

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SLIDE 48

T790M probe intensity WT probe intensity

LC7P1 2253:4537 33.2% 727/mL

T790M probe intensity WT probe intensity

LC7P3 1:9950 <LoD

T790M probe intensity WT probe intensity

LC7P5 10:9339 0.11% 4/mL

T790M probe intensity WT probe intensity

LC7P9 115:8688 1.3% 35/mL

5001000 1080 1140 1200 1260 1320 1380 1440 1500 0.01 0.1 1 10 100

T790M Allele Frequency (plasma)

Erlotinib Rociletinib (375mg) Rociletinib (250mg) 33.2% 0.11% 0.09% 1.3% Osimeritinib 0.04%

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25 30 35 40 400 600 800

T790M copies per mL plasma

727 4 3 35 2

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25

RECIST Score

Days on TKI therapy

  • 15.6%
  • 36.8%
  • 31.6%
  • 21.1%
  • 31.6%
  • 31.6%
  • 31.6%

Progession (non-RECIST lesions)

Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M)

Slide courtesy of Sabine Hellwig, PhD

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SLIDE 49

T790M probe intensity WT probe intensity

LC7P1 2253:4537 33.2% 727/mL

T790M probe intensity WT probe intensity

LC7P3 1:9950 <LoD

T790M probe intensity WT probe intensity

LC7P5 10:9339 0.11% 4/mL

T790M probe intensity WT probe intensity

LC7P9 115:8688 1.3% 35/mL

5001000 1080 1140 1200 1260 1320 1380 1440 1500 0.01 0.1 1 10 100

T790M Allele Frequency (plasma)

Erlotinib Rociletinib (375mg) Rociletinib (250mg) 33.2% 0.11% 0.09% 1.3% Osimeritinib 0.04%

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25 30 35 40 400 600 800

T790M copies per mL plasma

727 4 3 35 2

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25

RECIST Score

Days on TKI therapy

  • 15.6%
  • 36.8%
  • 31.6%
  • 21.1%
  • 31.6%
  • 31.6%
  • 31.6%

Progession (non-RECIST lesions)

Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M)

Slide courtesy of Sabine Hellwig, PhD

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SLIDE 50

T790M probe intensity WT probe intensity

LC7P1 2253:4537 33.2% 727/mL

T790M probe intensity WT probe intensity

LC7P3 1:9950 <LoD

T790M probe intensity WT probe intensity

LC7P5 10:9339 0.11% 4/mL

T790M probe intensity WT probe intensity

LC7P9 115:8688 1.3% 35/mL

5001000 1080 1140 1200 1260 1320 1380 1440 1500 0.01 0.1 1 10 100

T790M Allele Frequency (plasma)

Erlotinib Rociletinib (375mg) Rociletinib (250mg) 33.2% 0.11% 0.09% 1.3% Osimeritinib 0.04%

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25 30 35 40 400 600 800

T790M copies per mL plasma

727 4 3 35 2

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25

RECIST Score

Days on TKI therapy

  • 15.6%
  • 36.8%
  • 31.6%
  • 21.1%
  • 31.6%
  • 31.6%
  • 31.6%

Progession (non-RECIST lesions)

Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M)

Slide courtesy of Sabine Hellwig, PhD

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SLIDE 51

T790M probe intensity WT probe intensity

LC7P1 2253:4537 33.2% 727/mL

T790M probe intensity WT probe intensity

LC7P3 1:9950 <LoD

T790M probe intensity WT probe intensity

LC7P5 10:9339 0.11% 4/mL

T790M probe intensity WT probe intensity

LC7P9 115:8688 1.3% 35/mL

5001000 1080 1140 1200 1260 1320 1380 1440 1500 0.01 0.1 1 10 100

T790M Allele Frequency (plasma)

Erlotinib Rociletinib (375mg) Rociletinib (250mg) 33.2% 0.11% 0.09% 1.3% Osimeritinib 0.04%

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25 30 35 40 400 600 800

T790M copies per mL plasma

727 4 3 35 2

5001000 1080 1140 1200 1260 1320 1380 1440 1500 5 10 15 20 25

RECIST Score

Days on TKI therapy

  • 15.6%
  • 36.8%
  • 31.6%
  • 21.1%
  • 31.6%
  • 31.6%
  • 31.6%

Progession (non-RECIST lesions)

Current/Future Directions

NSCLC Serial Monitoring (EGFR T790M)

Slide courtesy of Sabine Hellwig, PhD

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SLIDE 52

G12V:WT 101:5571 1.8% 53 copies/mL 16-0004869 G G T G C

G12V detected

T G

NTC Healthy cfDNA ctDNA

Multiplex PCR products

NTC Healthy pool ctDNA

KRAS exon2 ICP products Qualitative analysis by Sanger Quantitative analysis by ddPCR:

Current/Future Directions

Pancreatic Cancer – KRAS exon 2 ice-COLD-PCR

Slide courtesy of Sabine Hellwig, PhD

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SLIDE 53

Current/Future Directions

Genotype/Phenotype Associations

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

Myelin Density Imaging Dynamic MRI

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SLIDE 54

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”

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SLIDE 55

Sabine Hellwig, PhD Keith Gligorich, PhD Mary Bronner, MD Carrie Fuertes, CRC Amy Hall, MB (ASCP) MLS (ASCP) David Nix, PhD Brett Milash, MS ARUP Brett Kennedy, PhD Daniel Baker, MS Elaine Gee, PhD Brendan O’Fallon, PhD Ashini Bolia, PhD

Acknowledgments

BMP-Core John O’Shea, PhD Katy Phillips Kevin Lee James Kline Andy Lee Funding

  • NIH K99CA168943
  • Department of Pediatrics research

support funds

  • Shameless begging

Randy Jensen, MD, PhD Howard Colman, MD, PhD