Subclonal phylogenetic structures in cancer revealed by ultra-deep sequencing.
During the clonal expansion of cancer from an ancestral cell with an initiating oncogenic mutation to symptomatic neoplasm, the occurrence of somatic mutations (both driver and passenger) can be used to track the on-going evolution of the neoplasm. All subclones within a cancer are phylogenetically related, with the prevalence of each subclone determined by its evolutionary fitness and the timing of its origin relative to other subclones. Recently developed massively parallel sequencing platforms promise the ability to detect rare subclones of genetic variants without a priori knowledge of the mutations involved. We used ultra-deep pyrosequencing to investigate intraclonal diversification at the Ig heavy chain locus in 22 patients with B-cell chronic lymphocytic leukemia. Analysis of a non-polymorphic control locus revealed artifactual insertions and deletions resulting from sequencing errors and base substitutions caused by polymerase misincorporation during PCR amplification. We developed an algorithm to differentiate genuine haplotypes of somatic hypermutations from such artifacts. This proved capable of detecting multiple rare subclones with frequencies as low as 1 in 5000 copies and allowed the characterization of phylogenetic interrelationships among subclones within each patient. This study demonstrates the potential for ultra-deep resequencing to recapitulate the dynamics of clonal evolution in cancer cell populations.
Campbell
Peter J
PJ
Wellcome Trust Sanger Institute, Hinxton CB10 1SA, United Kingdom.
Pleasance
Erin D
ED
Stephens
Philip J
PJ
Dicks
Ed
E
Rance
Richard
R
Goodhead
Ian
I
Follows
George A
GA
Green
Anthony R
AR
Futreal
P Andy
PA
Stratton
Michael R
MR
D000465:Algorithms; D001483:Base Sequence; D019070:Cell Lineage; D002999:Clone Cells; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000473:pathology*; D008969:Molecular Sequence Data; D009711:Nucleotides; D017422:Sequence Analysis, DNA / Q000379:methods*
D007143:Immunoglobulin Heavy Chains; D009711:Nucleotides