Accumulation of somatic mutations in proliferating T cell clones from children treated for leukemia

Abstract

There is continued controversy as to the sequential steps and mechanism(s) responsible for the in vivo acquisition of multiple mutations during neoplastic transformation. We investigated the in vivo clonality and mutational spectra of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations in T cells from children with acute lymphocytic leukemia (ALL) to gain insight into the mutagenic mechanisms associated with leukemogenesis. We observed several instances of multiple, independent HPRT mutations accumulating in vivo in T cell receptor (TCR) gene defined clones that had undergone extensive pre-and/or post-thymic expansion following chemotherapy. In addition, we also detected the accumulation of multiple unique single mutations within distinct expanding postthymic T cell clones. This pattern of clonally restricted hypermutability is compatible with extensive cell proliferation and selection alone without postulating genomic instability. These observations provide a paradigm for a continuum of cellular events that eventually results in the clonal accumulation of mutations in selected populations of cells in vivo and may provide insight into the primary genetic events associated with leukemogenesis, as well as the development of second malignancies and drug resistance following chemotherapy. Leukemia (2001Leukemia ( ) 15, 1898Leukemia ( -1905

Analysis of Genetic Alterations and Clonal Proliferation in Children Treated for Acute Lymphocytic Leukemia

“…We investigated the number of unique mutations in each subject sample by determining both the sequence of the TCRh CDR3 gene region as well as the specific HPRT mutation for each T cell HPRT mutant. This approach allows us to determine the extent of pre-or postthymic clonal proliferation, and allows calculation of the frequency of unique HPRT mutations (13). Analysis included 540 mutants (2-10 per sample) characterized from 71 blood samples from 42 subjects, in which some were represented in more than one phase of therapy.…”

“…We observed 15 examples of postthymic proliferation and 3 examples of ''proliferative'' clonality (ref. 13; same TCR and different HPRT mutations) from 12 of 38 children during treatment. During therapy, there were also six instances of clonality from four children in which the particular type could not be determined.…”

“…The rearrangement of the TCR genes in mutant isolates provides an independent measure of clonality and thus allows measurement of clonal proliferation that can be followed over time (13,14). The extent of clonal proliferation was determined by sequence analysis of the TCRh CDR3/ variable regions.…”

“…The PCR products were then run on an agarose gel, excised, purified, and sequenced. The TCRh CDR3 region sequence and the type of HPRT mutation(s) were used together to determine if mutant isolates were unique or of clonal origin, as previously described (13).…”

Genotoxicity of Therapeutic Intervention in Children with Acute Lymphocytic Leukemia

“…However, we do have the ability to address these questions by studying the relationship among HPRT-mutational spectra, cell proliferation, and selection through the use of TCR␤ gene analysis of each mutant isolate as an independent marker of clonality. We have previously used this relationship to demonstrate that in a subset of children treated for ALL, there is evidence of the emergence of both genomic instability and clonal proliferation (39,40). In the later study, we reported on the extent of clonal proliferation of HPRT-mutant isolates from 12 children treated for ALL who had Mfs Ͼ 30-fold higher than age-matched controls.…”