Download - Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

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Page 1: Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

TCGA-06-0214

TCGA-06-5415

TCGA-14-2554

TCGA-26-5132

EGFR vIII var. 1

EGFR vIII var. 2

EGFR vIII var. 3

EGFR C-ter deletion

EGFR vIII var. 1

EGFR vIII var. 2

EGFR vIII var. 1

Δ13-15

EGFR vIII var. 2

EGFR vIII var. 3

EGFR vIII var. 1

EGFR vIII var. 2

EGFR vIII var. 3

A

B

C

D

Supplemental Figure 1

Page 2: Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

Number of read pairs (million)10

010

110

20

0.5

1

Different blood normal libraries

Bulk primary tumor libraries

Down-sampled bulk tumor libraries

Selected single-cell WGA libraries

Poor WGA libraries

0.5

1

100

101

102

Number of read pairs (million)

KS

met

ric (

~60

Kb

bins

)K

S m

etric

(~

57 K

b bi

ns)

0 0.5 10

0.5

1

KS metric

% o

f pile

up r

eads

rem

oved

Down-sampled bulk tumor librariesSingle-cell WGA libraries

A

C

BBT340

BT325

0

Supplemental Figure 2

Page 3: Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

7: 54,560,250--55,510,789

p11.1

A

p12.1 p11.2

BT325

EGFR

p12.1 p11.1

7: 54,861,307--55,396,737

p11.2

B

C

D

EGFRCEP7

EGFR 12 13 14 15 16 Iso A (1210aa)

BT340

EGFR

Supplemental Figure 3

Chr. 7 Chr. 7

p12.1 p11.1p11.2

Two distinct deletions spanning ex. 14-15

Page 4: Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 161718

1920

2122

X

T/N

cop

y ra

tio

0.5

1

2

Subclonal

Homozygous deletion

Hemizygous deletion

Trisomic

Tetrasomic

Disomic

Clonal54

1

2

0.5

1.5

0

T/N

cop

y ra

tio

a

a. Subclonal deletions in chrs. 1, 4, 6

EGFR

CDKN2A/2B

BA Bulk copy number of BT325 Clonality analysis of SCNA in BT325

Supplementary Figure 4

Page 5: Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

AGermline heterozygous site

Reference baseAlternate base

Genotype

Possibe haplotypes

~1Kb

Cluster 1 Cluster 2

Clustering based on pairwise distance

Haplotype inference

Retained allelotypeDeleted allelotype

LOH populationHeterozygous population

RD

R R D R D

. . R R . . R

. . . . .. . . . .

1

2

3

4

5

6

Single-cell allelotyping

D D RD D R

. . . R . . R

R R . . R ..

D

0

0.5

1

Allelotype ratio (deleted vs. retained) within SNS

Allelotype distribution within two clusters

Cluster 1

Cluster 2

RD

RD

T/N

mix

dipl

oid

F Cell

B

A

Loss -of-heterozygosity

Haplotypes

Heterozygous

Genotypes from incompletesingle-nucleus sequencing

Homozygous loss

A

B

Genotype from completesingle-nucleus sequencing

1

2

3

4

5

6

Pairwise distance

2/3

C

cell 3 (LOH) vs.

cell 1(Het)

cell 3 (LOH) vs.

cell 4 (LOH)0

Pairwise comparison

E

G

0

0.01

0.02

0.03

0.04

0.05% of bases containing at least 1 error read avg: 0.019; std. dev: 0.005 % of reads containing at least 1 error base avg: 0.012; std. dev: 0.003

Tumor cells with chr. 10 deletion

H

Supplemental Figure 5

Page 6: Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

1

0.5

0

Haplotype ratio in Chr. 10 (Deleted vs. Retained)

Chromosome 7 copy ratio

Cells with less even coverageCells with more even coverage

0

1

2

3

Normal (diploid)

Tumor cells with complete 6q deletionTumor cells retaining multiple 6q segments

A

B

C

Chromosome 10 deletion

Chromosome 7 amplification

Chromosome 6 haplotypingChr. 9

haplotyping

D E

Haplotype compositions in regions with subclonal deletions

Retained haplotype Deleted haplotype

0 0.5 1 0 1000

Haplotype ratio

D:RTotal # of SNPs

53.6--57 Mb 63.3--66.1 Mb

27.1-30.9 Mb31.8-32.0 Mb

28--33 Mb

Clonal Events

Subclonal deletions in 6q and 9p Subclonal Chr. 16 loss

Subclonal Events

Supplemental Figure 6

0 1

Haplotype ratioD:R

Chr

. 16

LOH

Chr

. 16

non-

LOH

CDKN2A hom. lossChr.10 hem. loss

Deletions in Chrs. 6 and 9

Chr. 16 loss

Page 7: Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

A

Chr9: 9--35Mb

Retained allelotype Deleted allelotype

B

0 0.5 1

Haplotype ratio (Deleted vs. Retained)

0 0.5 1 1.5 2

Chr. 7 copy ratio

C9p deletion Chr. 10 deletion Trisomy 7

Haplotyping

Clonal Events

Chr7: 54560159-54560356 Chr7: 55510459-55510566

Tumor/normal mixture 1

Tumor/normal mixture 2

E

Amplicon 7: 54,560,250--55,510,789

F

Tumor/normal mixture 1

Tumor/normal mixture 2

Chr3: 22615037-22615438Chr9: 21029084-21029485

Presence of EGFR amplification in T/N mixtures

Presence of Chr. 9-Chr. 3 translocation in T/N mixtures

D

1B1A

8B8A

9B9A

Rearranged segments by the reference assembly

Rearranged segments in derivative chromosomes

Complex rearrangement disrupting CDKN2A

Bulk210

CN

RepresentativeSNS library

Pool ofSNS libraries

Chr. 1

Chr. 8

Chr. 9

Chr. 3 fragments

20,900--22,150Kb

CDKN2A/CDKN2B

9A 8B

1A 9B

8A 1B

der (1,9)

der (8,1)

der (9,8) × 2

Supplementary Figure 7

Page 8: Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

Chr. 2

Chr. 8

2:187389362

2:187389633

8:32908298

8:32909007

2:1892626238:2261740

2:1894072648:2261928A

8p-tel.

2q-tel.

8p-tel.

8:32908298

B-a5 • -a6 • -a1

a7 • -a3 • -a4 • a9 • a8 • -a2 • -a10

Chr. 12

a b c de f g12a • 12e • 12b • 12f • -12d • 12c • 12f

C Chr. 14

a

b

c d

e

f g nlkjih

m14a • -14l • 14g • -14f • 14d • 14c • 12f

? • 14i • -14j • 14k • 14h • ?

D

Chr. 9Chr. 6 9B6A

Simple deletion

Chr.6 Chr.9 CEP6

Chr. 9

20 25 30 35 40

E

Bulk CN50 60 7055 65

FISH probes Chr. 6Bulk CN

Chr.6 Chr.9 CEP9

extrachromosomal

amplicons

FISH probes Chr. 9chr6:71,726,042-71,726,935 chr9:27,138,441-27,140,176

+

connects to the Chr. 6 amplicon

connects to the Chr. 6 segment

connects to another Chr. 6 segment

connects to another Chr. 6 amplicon

Non-amplified Chr. 6 segment Non-amplified Chr. 9 segment

Chr. 6 amplicon Chr. 9 amplicon

Inferred derivative chromosome in cluster 1

9B6A

9B

Inferred derivative chromosome in cluster 2

6A 9B

6A

Chromothripsis

6.5Mb

9c

9b

6d

6c

6b6a

dm 6:9

Supplemental Figure 8

Page 9: Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

Chromosome 1 (Mb) Chromosome 4 (Mb) Chromosome 6 (Mb)

20 22 24 26 183 185 187 110 120 130 140 150 160

0.5

Min

or a

llele

fre

quen

cy

0

1

Bulk CN

0.50 1 1.5

All tumor nuclei

Haplotype ratio (Deleted vs. Retained)

Nuclei retaining heterozygosity

Nuclei having lostheterozygosity

Two nucleus mixture

A

Retained allelotype Deleted allelotype

0.5

01

1.5

Nuclei retaining heterozygosity

Nuclei having lostheterozygosity in chr. 1, 4, 6

1:1 mixture

00.

51

Chr. 10

Deletions in chrs 1, 4, 6

Tumor nuclei Non-tumor nuclei

CDKN2A homozygous deletionChr. 10 hemizygous deletion Heterozygous Chr. 10

Diploid normal 1:1 mixture T/N

Hap

loty

pe r

atio

D:R

Hap

loty

pe r

atio

D:R

Chromosome 1 (Mb) Chromosome 4 (Mb) Chromosome 6 (Mb)20 22 24 26 183 185 187 110 120 130 140 150 160

0.5

Min

or a

llele

fre

quen

cy

0

1

Bulk CN

LOH tumor nuclei only

1a 1b

1A4a 4b

4A4A4A

6a 6b 6c 6d 6e 6f 6h6g 6i

6D 6E 6F6G

Rearranged segments by the reference assembly

Rearranged segments in derivative chromosomes

E

Chr. 1

Chr. 4

Chr. 6

4a 4b

1a

6a

1b6i

6e6b

6c6f

6h

6g

der 1:6

der 4

der 6:1

6d

Supplementary Figure 9

F

D

B C

Page 10: Supplemental Figure 1 - Cancer Discovery · Supplemental Figure 6 0 1 Haplotype ratio D:R Chr. 16 LOH Chr. 16 non-LOH CDKN2A hom. loss Chr.10 hem. loss Deletions in Chrs. 6 and 9

0

50

100

150

0

50

100

150

100

50

150

0

0

50

100

150

Per

cent

Sur

viva

lP

erce

nt S

urvi

val

Per

cent

Sur

viva

lP

erce

nt S

urvi

val

C D

Erlotinib (nM)10 10 2 10 3 10 4

10 10 210 3 10 41

Lapatinib (nM)

0.01 0.1 101 101 100Neratinib (nM)Afatinib (nM)

vII

vIII

Vector + IL-3

WT + IL-3

EGFR vII EGFR vIII

IC50(nM) 4.37.8

vII

vIII

Vector + IL-3

WT + IL-3

EGFR vII EGFR vIII

IC50(nM) 930210EGFR vII EGFR vIII

IC50(nM) 8455

EGFR vII EGFR vIII

IC50(nM) 0.480.62

A B

Supplemental Figure 10: