CLUSTERING OF SOMATIC MUTATIONS TO CHARACTERIZE CANCER HETEROGENEITY WITH WHOLE GENOME SEQUENCING

J Becq1, A Alexa1, K Cheetham1, R Grocock1, Z Kingsbury1, A Timbs2, D McBride1, S Humphray1, M Ross1, A Schuh2 and D Bentley1
1illumina Cambridge Ltd.,

Chesterford Research Park, Cambridge, UK and 2Oxford NIHR Biomedical Research Centre, University of Oxford, Oxford UK

CANCER HETEROGENEITY
Founder mutations

TIME-SERIES ANALYSIS OF SOMATIC SINGLE NUCLEOTIDE VARIANTS

1. 2. 3. Select SNVs seen in at least one time-point Filter for good quality (not in copy number aberration region, 15x < coverage < 200x in all but 1, genotype Qscore > 15 in all but 1) Measure mutant Allele Frequency (mutant AF) at each time-point
Principal component analysis
REF Base = A Mutations resistant to treatment
A A A T T T T

PROPOSED CELL POPULATION

Founder mutations Early subclone mutations Late subclone mutations 13% cancer 8%
Trees containing this subclone are rejected because its frequency is below 3% at most (below noise level)

Cancers are genomically diverse and dynamic entities Clonal evolution generally selects for increased proliferation and survival, and might lead to invasion, metastasis and therapeutic resistance

Mutations sensitive to treatment

5%

2%

3%

Mutant AF: 4/7 = 0.57

3%

3%

47%

89%

95%

chr chr1 chr2 chr3 chr21 chr22 chrX

position

R1

P1

R2

preT3

Stage D Emerging mutations after treatment

154543705 0.0000 0.0000 0.2174 0.4255 0.4242 198266834 0.5000 0.6364 0.3478 0.4091 0.5938 31107645 1592215 32831696 0.0303 0.0000 0.1928 0.4146 0.4483 0.0000 0.0000 0.0476 0.0526 0.2500 0.4211 0.5294 0.0538 0.0000 0.0000

CLINICAL STUDY
DNA samples were collected from a patient with Chronic Lymphocytic Leukemia at different time-points during his treatment
Chlorambucil D Diagnosis GL Germline Tumour progression Remission Fludarabine Chlorambucil Rituximab R1 Relapse P1 Remission R2 Relapse preT3

Stage R1

A T T T T T T T T

Component 2

80% Non-cancer 4%

88%

12%

1%

0%

142716811 0.0000 0.0000 0.4737 0.9000 1.0000

Mutant AF: 8/9 = 0.89

1%

36%

8%

2%

R1

P1

4. Cluster SNVs with similar mutant AF profiles using k-means

R2

preT3

Component 1

VALIDATION WITH DEEP SEQUENCING

Ultra-deep sequencing (50,000x) of amplicons for all somatic SNVs with non-synonymous consequences Report accurate mutant allele frequency for each amplicon

short remission duration, aggressive disease, death despite treatment time

MUTATION PROFILES OF ALL SOMATIC SNVS

mutation of interest
Amplify by PCR Sequence on MiSeq instrument

Target

DETECTION OF TUMOUR SPECIFIC MUTATIONS

Germline
Mutations present at all stages, regardless of treatments Emerging mutations (mutant AF is 0% at early stages)

Black lines: somatic SNVs with non-synonymous or nonsense consequences

Fludarabine Chlorambucil Rituximab

Mutations decreasing after Fludarabine + Chlorambucil + Rituximab treatment

Expanding mutations (mutant AF is >0% at early stages)

Tumours

FROM CLUSTERS TO CLONES

Enumerate possible cells
possible common ancestor (founder clone)

Enumerate most parsimonious phylogenies

CONCLUSION
There are two late subclones, one present at diagnosis and one emerging after the second line of treatment
6% 9% 13% 5% 3%

Normal BAM

Somatic variants
realignment
Normal realigned reads

candidate indel search

Tumour BAM

Candidate Indels

realignment
w Tumour realigned reads Average HET mutant AF x 2
At each time-point

<1% x y z

44%

87%

94%

Deep-sequencing also provides accurate proportions

D
# of Mutational D SNVs group 1136 96% 686 80% 1241 3% 502 3% R1 99% 88% 3% 5% P1 64% 12% 47% 4% R2 92% 1% 89% 5% preT3 98% 0% 95% 5% w +x +y +z = 100% - GL contamination =

R1

P1

R2

preT3

Somatic Caller

+y

Post-call filtration

Time-series whole genome sequencing at 30x is sufficient to provide a representation of tumour cell populations / heterogeneity, provided each cell population is >10% Deep sequencing has confirmed the WGS analysis while providing greater sensitivity as mutations at very low frequencies (~1%) can be detected

Small Somatic variants

Because of constant < 10% mutant AF, the green mutational group is considered as noise

+y

+z