Mutagenesis vol. 19 no. 5 © UK Environmental Mutagen Society 2004; all rights reserved.
Analysis of microsatellite instability in children treated for acute lymphocytic leukemia with elevated HPRT mutant frequencies
1Department of Pediatrics, 2Department of Medical Biostatistics, 3Vermont Cancer Center and 4Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA
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Survival rates of children treated for cancer have increased dramatically over the last 25 years following the development of risk-directed multi-modality treatment protocols. As a result, there is a rapidly growing population of children and young adult cancer survivors in which the long-term genotoxic effects of chemotherapeutic intervention is unknown. We have previously observed that children treated for acute lymphocytic leukemia (ALL) have significantly increased somatic mutant frequencies (Mfs) (30- to 1300-fold higher) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene in their non-malignant peripheral T cells compared with children at diagnosis or controls. In order to gain insight into the etiology of the observed dramatic increase in Mfs following antineoplastic therapy, we investigated the prevalence of microsatellite instability (MSI), reflective of a defect in DNA mismatch repair (MMR), in children with ALL at diagnosis, during and after chemotherapy and compared them with healthy age-matched controls. MSI analysis using five microsatellite markers was performed on 167 T cell isolates from 40 healthy children and on 842 T cell isolates from 50 patients treated for ALL. High-frequency MSI (MSI-high) was identified in 2 healthy children (5%) and in 2 of 20 ALL subjects at the time of disease recurrence (relapse) (10%). There was no statistically significant difference between the prevalence of MSI-high in patients at the time of ALL relapse and healthy children, nor between the children with ALL at other time points and healthy children. These data indicate that MMR defects, represented by MSI, are not a significant contributor to the elevated HPRT Mfs seen in children treated for ALL. However, in a small number of patients chemotherapy may play a role in the selection of cells with defects in MMR that may have long-term clinical implications.
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Introduction |
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Acute lymphocytic leukemia (ALL) is the most common childhood malignancy. Since the early 1960s the overall 5 year survival rate for children with ALL has improved to between 80 and 90% following the development of national cooperative standardized multiple modality, risk of relapse-directed chemotherapeutic treatment protocols (Gloeckler et al., 1999
). A consequence of this success is that there is a growing population of pediatric cancer survivors in which the genotoxic effects of the chemotherapy is unknown. A potential adverse effect of this treatment is that these children acquire cellular and genetic alterations in non-tumor somatic cells that may contribute to the observed late effect clinical complications, including an increased risk for developing second malignant neoplasms (SMN) (Friedman and Meadows, 2002).
We have previously reported that children treated for ALL have significantly elevated somatic mutant frequencies (Mfs) (30- to 1300-fold higher) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene in their non-malignant peripheral T cells compared with children at diagnosis or controls (Finette et al., 2000). Of importance was that the HPRT Mfs remained elevated, even years after the completion of chemotherapy, while continuing to increase at a rate similar for normal age-matched controls (Rice et al., 2004). The precise genetic mechanisms associated with this dramatic increase in Mfs following chemotherapy and the potential role of this increased mutational load on the risk of SMN is unknown. Possible mechanisms for the increased Mf include proliferation defects that allow unregulated cell division that results in a higher spontaneous mutation rate or genomic instability caused by a defect in a DNA repair system. Resistance to several chemotherapy drugs has been attributed to a deficiency in DNA mismatch repair (MMR) and is thus a potential mechanism responsible for the observed elevated somatic HPRT Mfs (Lage and Dietel, 1999). MMR is a post-replicative repair system that ensures genome stability by fixing base–base mispairs and insertion/deletion loops (IDLs) that form from slippage of the primer against the template strand, particularly at simple repetitive sequences such as microsatellites. Microsatellite instability (MSI), defined as variations in microsatellite repeat length, is reflective of MMR deficiency. Defects in MMR result in an increased mutation rate and genomic instability. Human cell lines with MMR gene defects exhibit a 50- to 750-fold increased mutation rate at HPRT compared with cell lines proficient in MMR (Glaab and Tindall, 1997).
MSI, initially found in colorectal cancer, is also frequently seen in gastrointestinal and endometrial tumors (Aaltonen et al., 1993; Eshleman and Markowitz, 1995; Arzimanoglou et al., 1998). MSI is relatively rare in childhood ALL tumors at 
10% (Takeuchi et al., 1997; Reato et al., 1998). However, MSI rates are rather high in both adult therapy-related leukemia cells and myelodysplastic syndrome, ranging from 44 to 94% (Ben-Yehuda et al., 1996; Das-Gupta et al., 2001; Sheikhha et al., 2002). In addition, tumors from children with various secondary pediatric cancers were positive for MSI (Gafanovich et al., 1999). These studies provide evidence that chemotherapy for a primary cancer may cause mutations in DNA genome stability genes that leads to MSI and thus an increased risk for SMN.
In order to gain insight into the genetic mechanisms associated with the observed elevated HPRT Mfs in children who received chemotherapy for ALL, we compared the overall prevalence and allelic frequency of MSI, as a biomarker reflective of MMR deficiency. This was done using non-tumor T cell isolates from children with high Mfs during or after chemotherapy and comparing them with T cell isolates from healthy age-matched controls.
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Materials and methods |
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Patient sample collection
A total of 101 heparinized peripheral blood samples from 50 children with B lineage ALL, from oncology units at the University of Vermont and other participating Pediatric Oncology Group (POG)/Children's Oncology Group (COG) institutions, were collected at the time of diagnosis, during chemotherapy and following the completion of therapy for primary and relapsed ALL. We also utilized blood samples previously obtained from 40 healthy children, 0–16 years of age, from the general and subspeciality pediatric clinics of the Department of Pediatrics at the University of Vermont (Finette et al., 1994
). Informed consent was obtained from all subjects following procedures approved by the Committee on Human Research at the University of Vermont and participating institutions of the cooperative POG/COG. Determination of HPRT Mfs and the isolation of T cell mutant isolates from peripheral blood samples were performed as previously described (Finette et al., 1994, 2000). Mutant and wild-type isolates were expanded, pelleted and then frozen at –80°C for future analysis.
Microsatellite PCR and analysis
T cell isolate pellets of 10 000–50 000 cells were lysed in a 10 µl volume containing TE (10 mM Tris–HCl and 1 mM EDTA), 0.5% Tween 20, 0.5% Nonidet P40 and 0.1 mg/ml proteinase K in a Perkin Elmer Cetus 480 thermocycler at 56°C for 60 min, followed by 96° for 10 min. Cell lysates were used as genomic material for MSI PCR. Between 2 and 20 individual T cell isolates per blood sample from each subject were studied. Each non-tumor T cell isolate was analyzed for MSI using five microsatellite markers: D2S123, D5S346 and D17S250 (dinucleotide repeats); BAT25 and BAT26 (mononucleotide repeats) (Boland et al., 1998). For PCR amplification of each of the microsatellite markers, 1 µl of cell lysate (template DNA) was mixed with 0.6 µM forward and reverse primer, in a total reaction volume of 25 µl containing 50 µM dNTPs, 10 mM Tris–HCl (pH 8.3), 40 mM NaCl, 1.5 mM MgCl2, 0.2 mM spermidine and 0.25 U Platinum Taq polymerase (Invitrogen). In addition, for each PCR reaction set-up a control reaction using 60 ng purified DNA from 36X4 accessory cells was run. 36X4 cells are HPRT– lymphoblastoid cells that are irradiated with a 137Ce source (8000 rad) and added to the T cells in small numbers to stimulate growth. For the markers D2S123, BAT25 and BAT26, amplification was performed as follows: 94°C for 5 min; 8 cycles of 94°C for 20 s, 60°C for 20 s (–1°C per cycle), 72°C for 40 s; 35 cycles of 94°C for 20 s, 52°C for 20 s, 72°C for 40 s; a final 10 min extension at 72°C. For the markers D17S250 and D5S346, amplification was performed as follows: 94°C for 5 min; 8 cycles of 94°C for 20 s, 64°C for 20 s (–1°C per cycle), 72°C for 40 s; 35 cycles of 94°C for 20 s, 56°C for 20 s, 72°C for 40 s; a final 10 min extension at 72°C. The PCR products were analyzed using an ABI 310 Genetic Analyzer with GeneScan Analysis V 3.1 (PE Applied Biosystems, Foster City, CA) and Genotyper 2.0 (PE Applied Biosystems) software. Fragment analysis of the PCR products allowed determination of either expansions or reductions of the microsatellite repeats, indicating MSI. A T cell isolate was classified as demonstrating either high frequency microsatellite instability (MSI-high), defined by 
2 markers demonstrating instability, or low frequency microsatellite instability (MSI-low), when only 1 marker demonstrated instability. In some cases material was unavailable for us to make comparisons of constitutional microsatellite marker size in each subject. In these instances the MSI marker profiles of all T cell isolates from a patient were compared with one another and the predominant marker sizes were classified as constitutional.
Statistical analysis
Confidence intervals for MSI prevalence were based on the Poisson distribution. The prevalences of MSI among children with ALL and among healthy children were compared using Fisher's Exact Test.
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The frequency and prevalence of MSI were determined in 167 T cell isolates from 40 healthy children aged 0–16 years. Two children each had one T cell isolate exhibiting MSI-high. One child, 6 years of age, had a single isolate exhibiting MSI-high out of three tested. The other child, 13 years of age, had one isolate out of four tested exhibiting MSI-high. Thus, the prevalence of MSI in healthy children was 5.0% and their background microsatellite mutation per allele frequency was estimated to be 0.66–0.78%, which is
2-fold higher than the 0.29% seen in normal T cells from healthy adults (Table I) (Hackman et al., 1995). A range is given for the microsatellite mutation per allele frequency because at times it was not possible to determine if there was indeed MSI at the mononucleotide repeat markers.
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The frequency and prevalence of MSI were also determined in 842 T cell isolates from 101 blood samples from 50 children (1–19 years of age) treated for B lineage ALL. These samples were obtained at the time of diagnosis, as well as at multiple time points during chemotherapy and following the completion of chemotherapy, for primary and relapsed ALL. Thus, ALL patients are often represented in more than one patient group (Table I). Samples were not available at all time points for all subjects. For children with ALL, MSI-high was observed in four T cell isolates from two children (7 and 18 years of age) at the time of disease recurrence (ALL relapse). One patient, CS072, had a single isolate exhibiting MSI-high out of six tested, while the other patient, CS143, had three isolates out of 19 exhibiting MSI-high. All three isolates from patient CS143 showing MSI-high had exactly the same MSI size pattern for all of the five markers studied, indicating that the instability had a clonal origin. Figure 1 shows an example of microsatellite marker fragment analysis indicating MSI in patient CS072. Follow-up analysis of patient CS072 during relapse chemotherapy and after therapy showed that at neither time point were any T cell isolates with MSI detected. Patient CS143 was also tested during relapse chemotherapy and showed no evidence of MSI in peripheral T cells from this subject. This suggests that relapse chemotherapy eliminated the isolates with MSI. The prevalence of MSI in children at the time of ALL relapse was 10% (Table I). There was no statistical difference between the prevalence of MSI-high in patients at the time of relapse and healthy children (P = 0.665) nor between any of the other groups of children with ALL and the healthy children. The microsatellite mutation per allele frequency for the ALL at relapse group (1.24–1.29%) is about twice that seen in healthy children (0.66–0.78%). The mutation per allele frequency for all patients within the ALL group as a whole, including relapse, is 0.25–0.26%. A limitation of this study is that we could not perform statistical analysis on a per T cell isolate basis. Three of the T cell isolates with MSI-high in the relapse group were from one patient, demonstrating the lack of independence between isolates from the same person, but the rare occurrence of MSI-high precluded the use of statistical methods that would adjust for this correlation.
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Discussion |
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Overall, this study indicates that MSI, a biomarker reflective of a defect in MMR, does not explain the elevated HPRT Mfs seen in children treated for ALL. In all of the ALL patient groups tested, from diagnosis through chemotherapy and after therapy, the only group with T cell isolates exhibiting MSI-high were at the time of ALL relapse. However, there was no statistically significant difference between the prevalence of MSI-high in the ALL relapse group and healthy children. Of importance is that MSI is not a significant contributor to the observed elevated HPRT Mfs in patients who received chemotherapy for ALL, however, in a small number of patients chemotherapy may play a role in the selection of cells with defects in MMR that may have long-term clinical implications.
Children with ALL are treated with a variety of chemotherapy drugs based on their risk of relapse-directed protocols. They include vincristine, cyclophosphamide, an alkylating agent, L-asparaginase, corticosteroids and the antimetabolites cytosine arabinoside, methotrexate, 6-mercaptopurine and 6-thioguanine. Teniposide (VM-26) and the anthracycline daunomycin are also included in the treatment. Resistance to the antimetabolites 6-thioguanine and mercaptopurine has been attributed to a deficiency in MMR, as 6-thioguanine–thymine mispairs are not recognized and are able to persist in the genome (Lage and Dietel, 1999). The anthracycline daunomycin intercalates with DNA and inhibits topoisomerase II by preventing the resealing of DNA breaks and teniposide, an epipodophyllotoxin, reacts with and inhibits topoisomerase II through the formation of DNA double-strand breaks. It has been shown that MMR-deficient cells have resistance to both the anthracyline doxorubicicn and the epipodophyllotoxin etoposide and, while the mechanism is not fully understood, it is thought that the MMR system may detect the inhibited topoisomerase II–DNA cleavage complex or stabilize it (Lage and Dietel, 1999).
Other researchers have found high rates of MSI in both adult therapy-related leukemia (15 of 16 patients) and in secondary tumors from children (9 of 9 patients) (Ben-Yehuda et al., 1996; Gafanovich et al., 1999). Although their data are not directly comparable with ours because they studied tumor cells while we studied non-tumor cells, differences in the results may also have occurred because we used a different panel of microsatellite markers and a different methodology to assess MSI. In addition, none of their patients had B lineage ALL as their primary malignancy. Thus, there is a difference in the chemotherapy protocols and probably the etiology of the primary diseases that may effect the development of MSI.
We are currently investigating other potential mechanisms that may be responsible for the elevated HPRT Mfs seen in children treated for ALL. Specifically, we are studying the role of clonal proliferation by sequencing the T cell receptor ß CDR3/variable regions of the HPRT T cell isolates, as well as investigating other possible defects in DNA repair pathways. Understanding the mechanisms behind the elevated Mfs may lead to improvements in less genotoxic chemotherapy protocols and in determining the risk of second malignancies.
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Acknowledgments |
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We thank Sheara Billado RPN for assistance with obtaining blood samples and Jami Rivers and Terri Messier for technical assistance. This work was supported by the National Institute for Child Health and Human Development grant 1R29HD35309, grant 1003312 from the Burroughs Wellcome Fund, National Cancer Institute grants 1K01CA77737, 1R01CA09094013 and P30CA22435 to the University of Vermont Cancer Center DNA Analysis Facility, and the Vermont Genetics Network through NIH grant 1P20RR16462 from the BRIN Program of the National Center for Research Resources.
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5 To whom correspondence should be addressed at: Department of Pediatrics, E203 Given Medical Building, University of Vermont, 89 Beaumont Avenue, Burlington, VT 05405, USA. Tel: +1 802 656 2296; Fax: +1 802 656 2077; Email: barry.finette{at}uvm.edu
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Received on June 14, 2004; revised on August 5, 2004; accepted on August 6, 2004.
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