Beruflich Dokumente
Kultur Dokumente
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
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
5%
2%
3%
3%
3%
47%
89%
95%
position
R1
P1
R2
preT3
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%
1%
36%
8%
2%
R1
P1
R2
preT3
Component 1
mutation of interest
Amplify by PCR Sequence on MiSeq instrument
Target
Tumours
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
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%
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
Because of constant < 10% mutant AF, the green mutational group is considered as noise
+y
+z