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

Abstract

The development of risk-directed treatment protocols over the last 25 years has resulted in an increase in the survival rates of children treated for cancer. As a consequence, there is a growing population of pediatric cancer survivors in which the long-term genotoxic effects of chemotherapy is unknown. We previously reported that children treated for acute lymphocytic leukemia have significantly elevated somatic mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in their peripheral T cells. To understand the molecular etiology of the increase in mutant frequencies following chemotherapy, we investigated the HPRT mutation spectra and the extent of clonal proliferation in 562 HPRT T cell mutant isolates of 87 blood samples from 47 subjects at diagnosis, during chemotherapy, and postchemotherapy. We observed a significant increase in the proportion of CpG transitions following treatment (13.6-23.3%) compared with healthy controls (4.0%) and a significant decrease in V(D)J-mediated deletions following treatment (0-6.8%) compared with healthy controls (17.0%). There was also a significant change in the class type percentage of V(D)Jmediated HPRT deletions following treatment. In addition, there was a >5-fold increase in T cell receptor gene usagedefined mean clonal proliferation from diagnosis compared with the completion of chemotherapeutic intervention. These data indicate that unique genetic alterations and extensive clonal proliferation are occurring in children following treatment for acute lymphocytic leukemia that may influence long-term risks for multifactorial diseases, including secondary cancers. (Cancer Res 2006; 66(17): 8455-61)

Mutagenicity and Potential Carcinogenicity of Thiopurine Treatment in Patients with Inflammatory Bowel Disease

“…Nineteen percent of the randomly selected HPRT mutants from IBD subjects remained uncharacterized, which is consistent with previous human biomonitoring HPRT spectra studies (36, 42). Reasons for the uncharacterized mutants include: 1) exon exclusions in mutants from female subjects in which splice alterations and deletions could not be confirmed or detected; 2) no cDNA detected, with genomic PCR products of exons 1 and 9 showing no mutations; 3) cDNA revealed no mutation, and 4) incomplete analysis.…”

“…The HPRT biomarker system also allows for the analysis of mutation spectra changes associated with exogenous/iatrogenic exposures, as well as the identification of independent in vivo sequential mutation events (34) and mutant clonal proliferations by simultaneously analyzing two independent clonality indicators, HPRT mutations and unique T cell receptor β ( TCR β ) CDR3 variable gene region sequences (35, 36). Experimental approaches, methods and interpretations of mutation spectra, and comparison of HPRT mutations and TCRβ CDR3 variable gene regions of mutant isolates for determining mutant proliferation have been previously described (34-36).…”

“…Experimental approaches, methods and interpretations of mutation spectra, and comparison of HPRT mutations and TCRβ CDR3 variable gene regions of mutant isolates for determining mutant proliferation have been previously described (34-36). HPRT mutation spectra were determined by analyzing up to 10 mutants per TP treated subject and up to 5 mutants per non-TP treated subject.…”

V(D)J Recombinase-Mediated TCR β Locus Gene Usage and Coding Joint Processing in Peripheral T Cells during Perinatal and Pediatric Development

“…Our laboratory and other investigators also showed that exposure to genotoxic agents, including pesticides, alkylating agents, and topoisomerase II inhibitors, as well as transplacental exposure to environmental tobacco smoke, can increase the frequency and alter the recombination site distribution of V(D)J-mediated recombinase events at cRSS (16,20,(40)(41)(42)(43)(44). Investigations at both nonimmune cRSS and immune RSS provide unique insight into developmental, gender, and environmental effects on the frequency and distribution of V(D)J recombinase-mediated rearrangements, coding joint processing, specificity, and the generation of T cell diversity.…”

“…We performed multiplex PCR analysis on cDNA to amplify the TCRb CDR3/V region using a mix of 26 TCR-Vb sense primers with a consensus Cb anti-sense primer, as previously described (19)(20)(21). PCR products were identified and isolated from 1.4% agarose gels and purified using QIAquick-spin columns (QIAGEN).…”