XPD Lys751Gln polymorphism in the etiology and outcome of childhood acute myeloid leukemia: a Children's Oncology Group report

DNA is continuously damaged by endogenous and exogenous mutagens. Repair of DNA damage is a complex process carried out by an array of DNA repair pathways, including nucleotide excision and base excision repair pathways. The nucleotide excision repair (NER) pathway eliminates the widest variety of damage to the human genome, including UV-induced photoproducts, bulky monoadducts, cross-links, and oxidative damage.¹ 

Hereditary genetic defects in DNA repair lead to increased risk of cancer. Individuals with xeroderma pigmentosum (XP), a rare autosomal recessive disease resulting from a defect in NER of UV-damaged DNA, have a 1000-fold increased risk of skin cancer.²  Cell-fusion analyses have identified 7 genetic complementation groups (XPA to XPG) that encode for proteins participating in different steps of the NER pathway.^3,4  Xeroderma pigmentosum complementation group D (XPD) is a major participant in NER pathway and is also involved in transcription initiation, control of cell cycle, and apoptosis.⁵ 

XPD functions as an evolutionarily conserved ATP-dependent helicase within the multisubunit transcription repair factor complex, TFIIH.^6-8  TFIIH has 2 distinct roles, first in basal transcription carried out by RNA polymerase II and second in NER of DNA damage. It appears that XPD protein needs to be present to maintain the stability of the TFIIH complex. XPD possesses both single-strand DNA-dependent ATPase and 5′-3′ DNAhelicase activities and is thought to participate in DNA unwinding during NER and transcription.^9,10  Mutations in the XPD gene can completely prevent DNA opening and dual incision, steps that lead to the repair of DNA adducts.¹¹ 

Genetic polymorphisms result in interindividual variation in DNA repair capacity and may, in part, account for differences in susceptibility of a cell to genotoxic agents and to malignancy.^12,13  Several single nucleotide polymorphisms, including an adenine (A) to cytosine (C) (A → C) transition, which leads to Lys751Gln in exon 23 of the XPD gene, have been shown to be associated with elevated frequency of chromosomal aberrations and a variety of environmentally induced cancers in adults.^14-17  There is also evidence that dysregulation of DNA repair proteins and NER pathways may be involved in pathogenesis and prognosis of some myeloid leukemia.

Recent data from elderly acute myeloid leukemia (AML) patients treated on MRC11 trial showed reduced event-free survival (EFS) and overall survival (OS) along with increased risk of developing treatment-related leukemia in XPD751 glutamine homozygotes (CC), suggesting that the glutamine variant confers greater protection against chemotherapy-induced leukemic blast-cell death, leading to earlier disease relapse and ultimately shorter overall survival.¹⁸  In this study we show that, in contrast to the adult data, XPD751 does not influence outcome of therapy in children with AML.

The study population included 456 children with de novo AML treated on Children's Cancer Group (CCG) therapeutic studies CCG-2941 (n = 37) and CCG-2961 (n = 419) between 1995 and 2002. Clinical data, including age, sex, white blood cell (WBC) count at diagnosis, race, presence of chloroma, presence of central nervous system (CNS) disease, and immunophenotype were collected prospectively (Table 1). Cases were classified on the basis of criteria established and revised by the French-American-British (FAB) Cooperative Study Group by central pathology review. All FAB categories except acute promyelocytic leukemia (APL-AML M3) were eligible for enrollment and were treated with the same chemotherapy regimens.

We randomly selected 578 healthy blood donors to determine control genotype frequencies: 432 were white and 146 were black controls.

CCG-2961 study was a randomized phase 3 trial of intensively timed induction, consolidation, and intensification therapy for pediatric patients with previously untreated AML or myelodysplastic syndrome (MDS).¹⁹  The study was conducted between August 1996 and December 2002. CCG-2941 was a feasibility pilot of the same chemotherapy regimen that preceded the randomized study. Induction included 5 drugs: idarubicin, etoposide, dexamethasone, cytarabine, and 6-thioguanine (IDA DCTER) given on days 0 to 3 followed by 5 drugs (daunorubicin, etoposide, dexamethasone, Ara-C, and 6-thioguanine) (DCTER) given on days 10 to 13.²⁰  Upon recovery of white blood cell and platelet counts, patients were randomly assigned to consolidation therapy consisting of the same sequence of drugs or to fludarabine/cytarabine/idarubicin. Intrathecal cytarabine was used for CNS prophylaxis. Patients with matched-related donors were assigned to allogeneic marrow transplant intensification. Pretransplant cytoreduction was busulfan and cyclophosphamide. Patients without a related donor received high-dose cytarabine/L-asparaginase (Capizzi II) and additional intrathecal cytarabine. After recovery from chemotherapy, patients were randomized again to either receive interleukin-2 or standard follow-up care. Patients who received transplants were not eligible for randomization to interleukin-2.

DNA extracted from diagnostic marrow samples using standard methods was normalized to 10 ng/μL. Genotyping was performed using a fluorescence-based allelic discrimination assay (TaqMan; Applied Biosystems, Foster City, CA). Gene-specific polymerase chain reaction (PCR) primers and fluorogenic probes for allelic discrimination are described in Table 2.

PCR cycling reactions were performed in 96-well microtiter plates in a GeneAmp PCR System 9600 (Perkin-Elmer). For each 25-μL reaction, 10 ng DNA template was added to the reaction mixture containing wild-type VIC and variant FAM probe, PCR mastermix (Applied Biosystems), and forward and reverse primers (final concentration 0.3 μM). Thermocycling was performed with an initial 50°C incubation for 2 minutes followed by a 10-minute incubation at 95°C. A 2-step cycling reaction was performed for 40 cycles with denaturation at 95°C for 15 seconds, and annealing and extension at 62°C for 1 minute. Results were analyzed by the automated TaqMan allelic discrimination assay using sequence detection system 2.1 software (ABI TaqMan 7700, Applied Biosystems).

DNA from healthy controls was extracted using standard techniques and genotyped as described for cases. Genotyping results were duplicated in 10% of samples; concordance between repeats was 100%. Furthermore, 10% of the samples also were genotyped using direct sequencing; concordance with TaqMan genotyping was 100%.

Data were analyzed from CCG-2941 and CCG-2961 through April 2005 for both studies. The significance of observed differences in proportions was tested using the Chi-square test and Fisher exact test when data were sparse. The Mann-Whitney test was used to determine the significance between differences in medians.²¹  The Kaplan-Meier method was used to calculate estimates of OS, EFS, and disease-free survival (DFS).²²  Estimates are reported with their Greenwood standard errors.²³  Differences in these estimates were tested for significance using the log-rank statistic.²⁴  OS is defined as time from study entry to death from any cause. EFS is defined as time from study entry to failure at the end of 2 courses, relapse, or death from any cause. DFS is defined as time from the end of one course of therapy to failure at the end of 2 courses, relapse, or death from any cause. Cumulative incidence estimates were used to determine relapse rate (RR) and treatment-related mortality (TRM). RR is defined as time from the end of one course of therapy to failure at the end of 2 courses, relapse, or death from progressive disease where deaths from nonprogressive disease were competing events. TRM is defined as time from study entry to death from nonprogressive disease where failures at the end of 2 courses, relapses and deaths from progressive disease were competing events. Differences between RR or TRM estimates were tested for significance using Gray test.²⁵  Children lost to follow-up were censored at their date of last known contact or at a cutoff 6 months prior to April 2005. Cox regression was used for multivariate models that looked at differences between groups adjusting for study assignment, age, sex, race, and WBC count.

Allele and genotype frequencies for cases and controls are shown in Table 3. XPD genotype frequencies for black and white controls were significantly different (P < .001). Genotype frequencies did not differ by sex (data not shown). Comparison of genotype frequencies was therefore performed separately for white cases versus white controls and black cases versus black controls. Genotype frequencies were similar in white cases and controls (Table 3; P = .125) and black patients and black controls (P = .239). The distribution of XPD genotypes was consistent with the Hardy-Weinberg equilibrium. Stratification of cases by age at diagnosis (0-2 y vs > 2-10 y vs > 10 y), WBC count at diagnosis (< 50 000 vs ≥ 50 000), AML subtype, or cytogenetics revealed no difference in genotype frequencies. There were more XPD751AC patients compared to AA in hyperdiploid cases; however, this difference should be interpreted cautiously due to the very small total number of patients with hyperdiploidy.

There were no significant differences in OS from study entry between patients with XPD751AA versus 751AC versus 751CC genotypes (53% ± 8% vs 50% ± 8% vs 45% ± 13% respectively at 5 years; log-rank P = .82; Figure 1). In addition, induction of remission did not vary by genotype (Table 1). When OS from end of one course of therapy for patients in remission was compared between different genotypes, there was no statistical difference between polymorphism subgroups; 57% ± 8% in XPD751AA versus 58% ± 8% in AC versus 52% ± 15% in CC patients at 5 years (log-rank P = .96).

Analysis of EFS from study entry in different genotypes also showed similar estimates: 41% ± 7% in XPD751AA versus 38% ± 7% in AC versus 41% ± 13% in CC patients at 5 years (log-rank P = .78) (Figure 2). DFS from the end of one course of therapy for patients in remission also was similar among the 3 genotypes (P = .78). Multivariate analyses that adjusted for study assignment, age, gender, race, and WBC count also suggested XPD genotypes did not have different OS or EFS.

There was no difference in TRM from study entry between different genotypes (18% ± 6% in AA vs 16% ± 5% in AC vs 12% ± 8% in CC patients at 5 years; P = .43) (Figure 3). RRs from the end of one course of therapy for patients in remission were also similar: 40% ± 8% in XPD751AA versus 42% ± 8% in 751AC versus 39% ± 14% in 751CC patients at 5 years (P = .92) (Figure 4). Thus, XPD genotype was not significantly associated with either resistant disease or treatment-related toxicity.

Pharmacogenetic polymorphism and variations in response to damage induced by chemotherapy are being intensively investigated as causes of differential susceptibility to leukemogenesis and differential response to therapy.^26-28  When investigating the clinical consequences of human polymorphism, it is important to target polymorphisms that likely change protein function and occur at significant frequencies in the population. Spitz et al²⁹  studied the functional consequences of the XPD Lys751Gln polymorphism among lung cancer patients and healthy controls. They reported that the variant Gln751Gln genotype was consistently associated with the suboptimal DNA repair capacity (DRC). This was determined by assessing the ability of host cells to remove DNA adducts induced by benzo(a)pyrene, a major constituent of tobacco smoke. This association was statistically significant among the lung cancer cases but not among the healthy controls, indicating a role for XPD in tobacco-related cancers. These data are controversial, however, and others such as Duell et al and Moeller et al reported no significant relationship between XPD Lys751Gln polymorphism and DNA repair proficiency.^30,31  These conflicting results have led to suggestions that functionality of the codon 751 polymorphism may be exposure—and pathway—specific, affecting both DNA repair and cell death.³²  Consistent with a role for XPD in cell death, P53-mediated apoptosis is attenuated in XPD mutated fibroblasts.^33,34  Furthermore, P53 interacts directly with the carboxy terminus of XPD, which includes the polymorphic codon 751 residue.³² 

A previous report of adult subjects showed that XPD751 genotype influenced susceptibility to therapy-related leukemia, but not de novo AML.¹⁸  In agreement with this, in this pediatric study we found no influence of XPD751 genotype on susceptibility to de novo AML in children. It should be noted that the present pediatric study focused on de novo and not therapy-related AML.

There is evidence that DNA repair (NER) protects against mutagenicity and toxicity by removing deleterious DNA lesions from the genome, including those induced by chemotherapy.^35-38  Because increased DNA repair plays an important role in resistance to platinum-based compounds, Park et al evaluated the effect of XPD Lys751Gln polymorphism on outcome of 73 patients treated with 5-fluorouracil (5-FU)/oxaloplatin for metastatic colorectal cancer. Their results showed a significant association between response to 5-FU/oxaloplatin and the XPD Lys751Gln polymorphism. Patients with Lys/Lys genotype had the longest median survival, and those with Gln/Gln genotype were 6 to 12 times more likely to have progressive disease.³⁹ 

Allan et al evaluated the association of XPD Lys751Gln polymorphism with outcome following chemotherapy for AML in 341 elderly patients (> 60 years of age) entered into the United Kingdom Medical Research Council (MRC) AML 11 trial.¹⁸  In this study XPD751 glutamine homozygotes had significantly inferior DFS at 1 year compared with patients with other genotypes. The authors postulated 2 general mechanisms by which the XPD codon 751 variant may modulate myeloid-cell death in response to chemotherapy: either via a direct role for XPD in signaling cell death or indirectly via XPD repair of protoxic DNA lesions.

In contrast to the findings of Allan et al,¹⁸  our study did not demonstrate any differences in outcome of AML therapy in children with different XPD751 genotypes. It is possible that children with AML differ from adults in terms of the biology of their disease, for example, studies show that older adults have increased frequency of adverse cytogenetic features compared to children.^40,41  Also, over time adult patients have more opportunity to accumulate additional genetic insults (secondary hits), with perhaps increased susceptibility to develop cancers that are more resistant to therapy. Outcomes for treatment of adult AML are commonly inferior to those reported in pediatric series.^42-45  Also, association studies involving genetic polymorphisms need to be interpreted cautiously in the context of differences in study or population variables. For example, compared to children, older adults have poorer tolerance of combination chemotherapy regimens, leading to the use of less-intensive treatment protocols, as well as increased levels of primary drug resistance associated with overexpression of P-glycoprotein (P-gp). Adults in the study by Allan et al¹⁸  received chemotherapy agents (daunomycin, cytosine arabinoside, thioguanine, etoposide) broadly similar to those received by the children in our study, but it is possible that the effect of XPD polymorphism is regimen specific. A subset of patients in the adult study received an alkylating agent (cyclophosphamide) in postremission therapy in contrast to our pediatric study, and half were randomized to interferon-α, either of which may have influenced the results. Progress in AML therapy in children has largely been made by aggressive intensification of chemotherapy. It is also possible that intensification of chemotherapy in children can overcome a marginal modulating effect of XPD genotype. Additionally, this XPD variant may be of functional importance in children with AML when combined with other XPD variants or polymorphic alleles of other DNArepair genes. Mechanistic studies further defining the functionality of XPD polymorphisms will help clarify the importance of variants at this site.

COG Active Institutions, listed alphabetically, are as follows: A.B. Chandler Medical Ctr, University of Kentucky, Lexington, KY; Advocate Hope Children's Hospital, Oak Lawn, IL; Albany Medical Center, Albany, NY; Alberta Children's Hospital, Calgary, AB, Canada; All Children's Hospital, St. Petersburg, FL; Allan Blair Cancer Centre, Regina, SK, Canada; Atlantic Health System, Morristown, NJ; Backus Children's Hospital at MHUMC, Savannah, GA; Baptist Children's Hospital, Miami, FL; Baystate Medical Center, Springfield, MA; Boston Floating Hospital for Infants & Children, Boston, MA; British Columbia's Children's Hospital, Vancouver, BC, Canada; Brookdale Hospital Medical Center, Brooklyn, NY; Brooklyn Hospital Center, Brooklyn, NY; Broward General Medical Center, Ft. Lauderdale, FL; C.S. Mott Children's Hospital, Ann Arbor, MI; Cabell Huntington Hospital, Huntington, WV; Cancer Research Center of Hawaii, Honolulu, HI; CancerCare Manitoba, Winnipeg, MB, Canada; Cardinal Glennon Children's Hospital, St. Louis, MO; Carilion Medical Center for Children at Roanoke Community Hospital, Roanoke, VA; Carolinas Medical Center, Charlotte, NC; Cedars-Sinai Medical Center, Los Angeles, CA; Centre Hospitalier Universitaire de Quebec, Ste-Foy, QC, Canada; Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, QC, Canada; Children's Healthcare of Atlanta, Emory University, Atlanta, GA; Children's Hem/Onc Team at Covenant Children's Hosp, Lubbock, TX; Children's Hospital and Regional Medical Center, Seattle, WA; Children's Hospital Central California, Madera, CA; Children's Hospital of Austin, Austin, TX; Children's Hospital of Eastern Ontario, Ottawa, ON, Canada; Children's Hospital of Michigan, Detroit, MI; Children's Hospital of Pittsburgh, Pittsburgh, PA; Children's Hospital of the Greenville Hospital System, Greenville, SC; Children's Hospital San Diego, San Diego, CA; Children's Medical Center Dayton, Dayton, OH; Children's Memorial Medical Center at Chicago, Chicago, IL; Children's National Medical Center—D.C., Washington, DC; Children's of New Orleans/LSUMC CCOP, New Orleans, LA; Childrens Hospital & Clinics Minneapolis & St Paul, Minneapolis, MN; Childrens Hospital Los Angeles, Los Angeles, CA; Childrens Hospital Medical Center Cincinnati, Cincinnati, OH; Childrens Hospital Medical Center-Akron, Ohio, Akron, OH; Childrens Hospital Oakland, Oakland, CA; Childrens Hospital of Orange County, Orange, CA; Childrens Hospital of Philadelphia, Philadelphia, PA; Childrens Hospital of Western Ontario, London, ON, Canada; Childrens Hospital-King's Daughters, Norfolk, VA; Childrens Memorial Hospital of Omaha, Omaha, NE; Christiana Care Health Services/A.I. duPont Inst., Wilmington, DE; City of Hope National Medical Center, Duarte, CA; Columbia Presbyterian College of Phys & Surgeons, New York, NY; Columbus Children's Hospital, Columbus, OH; Connecticut Children's Medical Center, Hartford, CT; Cook Children's Medical Center, Fort Worth, TX; Dana-Farber Cancer Institute and Children's Hosp, Boston, MA; Dartmouth-Hitchcock Medical Center, Lebanon, NH; DeVos Children's Hospital, Grand Rapids, MI; Doernbecher Childrens Hospital—OHSU, Portland, OR; Driscoll Children's Hospital, Corpus Christi, TX; Duke University Medical Center, Durham, NC; East Carolina University School of Medicine, Greenville, NC; East Tennessee Childrens Hospital, Knoxville, TN; East Tennessee State University, Johnson City, TN; Eastern Maine Medical Center, Bangor, ME; Emanuel Hospital-Health Center, Portland, OR; Florida Hospital Cancer Institute, Orlando, FL; Geisinger Medical Center, Danville, PA; Georgetown University Medical Center, Washington, DC; Gundersen Lutheran, La Crosse, WI; Hackensack University Medical Center, Hackensack, NJ; Hopital Sainte-Justine, Montreal, QC, Canada; Hospital for Sick Children, Toronto, ON, Canada; Hurley Medical Center, Flint, MI; Indiana University—Riley Childrens Hospital, Indianapolis, IN; Inova Fairfax Hospital, Fairfax, VA; IWK Health Centre, Halifax, NS, Canada; Janeway Child Health Center, St. John's, NF, Canada; Joe DiMaggio Children's Hospital at Memorial, Hollywood, FL; John Hunter Children's Hospital, Newcastle, NSW, Australia; Johns Hopkins Hospital, Baltimore, MD; Kaiser Permanente Medical Group, Inc., Northern CA, Sacramento, CA; Kalamazoo Center for Medical Studies, Kalamazoo, MI; Kingston General Hosp/Kingston Regional Cancer, Kingston, ON, Canada; Kosair Childrens Hospital, Louisville, KY; Loma Linda University Medical Center, Loma Linda, CA; Loyola University Medical Center, Maywood, IL; Lutheran General Childrens Medical Center, Park Ridge, IL; M.D. Anderson Cancer Center, Houston, TX; Madigan Army Medical Center (USOC), Tacoma, WA; Maimonides Medical Center, Brooklyn, NY; Maine Children's Cancer Program, Scarborough, ME; Marshfield Clinic, Marshfield, WI; Mary Bridge Hospital, Tacoma, WA; Massachusetts General Hospital, Boston, MA; Mayo Clinic and Foundation, Rochester, MN; McGill Univ Health Ctr - Montreal Children's Hosp, Montreal, QC, Canada; McMaster University, Hamilton, ON, Canada; Medical College of Georgia Childrens Medical Ctr, Augusta, GA; Medical University of South Carolina, Charleston, SC; Memorial Sloan Kettering Cancer Center, New York, NY; Mercy Children's Hospital, Toledo, OH; MeritCare Medical Group DBA Roger Maris Cancer Ctr, Fargo, ND; Methodist Children's Hospital of South Texas, San Antonio, TX; Miami Children's Hospital, Miami, FL; Michigan State University, East Lansing, MI; Midwest Children's Cancer Center, Milwaukee, WI; Miller Children's Hospital/Harbor-UCLA, Long Beach, CA; Mission Hospitals, Asheville, NC; Montefiore Medical Center, Bronx, NY; Mount Sinai Medical Center, New York, NY; Mountain States Tumor Institute, Boise, ID; Naval Medical Center/Portsmouth (USOC), Portsmouth, VA; Nemours Children's Clinic-Jacksonville, Jacksonville, FL; Nemours Children's Clinic-Orlando, Orlando, FL; Nevada Cancer Research Foundation—CCOP, Las Vegas, NV; New York Hospital-Cornell Univ Medical Center, New York, NY; New York Medical College, Valhalla, NY; New York University Medical Center, New York, NY; Newark Beth Israel Medical Center, Newark, NJ; North Texas Hosp for Children at Med City Dallas, Dallas, TX; Ochsner Clinic, New Orleans, LA; Penn State Children's Hospital, Hershey Med Ctr, Hershey, PA; Phoenix Childrens Hospital, Phoenix, AZ; Presbyterian Hospital, Charlotte, NC; Presbyterian/St Lukes Medical Center and CHOA, Denver, CO; Primary Childrens Medical Center, Salt Lake City, UT; Princess Margaret Hospital for Children, Perth, WA., Australia; Rainbow Babies and Childrens Hospital, Cleveland, OH; Raymond Blank Children's Hospital, Des Moines, IA; Rhode Island Hospital, Providence, RI; Roswell Park Cancer Institute, Buffalo, NY; Royal Children's Hospital, Brisbane, Herston, Brisbane, QLD, Australia; Royal Children's Hospital, University of Melbourne, Parkville, VI, Australia; Rush-Presbyterian St. Luke's Medical Center, Chicago, IL; Sacred Heart Children's Hospital, Spokane, WA; Sacred Heart Hospital, Pensacola, FL; Saint Barnabas Medical Center, Livingston, NJ; Saint Peter's University Hospital, New Brunswick, NJ; San Jorge Children's Hospital, Santurce, PR; Santa Barbara Cottage Children's Hospital, Santa Barbara, CA; Saskatoon Cancer Center, Saskatoon, SK, Canada; Schneider Children's Hospital, New Hyde Park, NY; Scott & White Memorial Hospital, Temple, TX; Sinai Hospital of Baltimore, Baltimore, MD; Sioux Valley Children's Specialty Clinics, Sioux Falls, SD; South Carolina Cancer Center, Columbia, SC; South Island Child Cancer Service, Christchurch, New Zealand; Southern California Permanente Medical Group, Downey, CA; Southern Illinois University School of Medicine, Springfield, IL; St John Hospital and Medical Center, Grosse Point Woods, MI; St. Christopher's Hospital for Children, Philadelphia, PA; St. Joseph's Hospital and Medical Center, Paterson, NJ; St. Jude Children's Research Hospital Memphis, Memphis, TN; St. Jude Midwest Affiliate, Peoria, IL; St. Mary's Hospital, West Palm Beach, FL; St. Vincent Children's Hospital—Indiana, Indianapolis, IN; St. Vincent Hospital—Wisconsin, Green Bay, WI; Stanford University Medical Center, Palo Alto, CA; Starship Children's Hospital, Auckland, New Zealand; State University of New York at Stony Brook, Stony Brook, NY; Stollery Children's Hospital, Edmonton, AB, Canada; SUNY Health Science Center at Brooklyn, Brooklyn, NY; SUNY Upstate Medical University, Syracuse, NY; Sutter Medical Center, Sacramento, Sacramento, CA; Swiss Pediatric Oncology Group Bern, Bern, CH, Switzerland; Swiss Pediatric Oncology Group Geneva, Geneva, CH, Switzerland; Swiss Pediatric Oncology Group Lausanne, Lausanne, CH, Switzerland; Sydney Children's Hospital, Randwick, NSW, Australia; T.C. Thompson Children's Hospital, Chattanooga, TN; Tampa Children's Hospital, Tampa, FL; Texas Children's Cancer Center at Baylor College of Medicine, Houston, TX; Texas Tech UHSC - Amarillo, Amarillo, TX; The Children's Hospital—Denver, CO, Denver, CO; The Children's Hospital at The Cleveland Clinic, Cleveland, OH; The Children's Hospital at Westmead, Westmead, NSW, Australia; The Children's Hospital of Southwest Florida Lee Memorial Health Sy, Ft. Myers, FL; The Children's Mercy Hospital, Kansas City, MO; The University of Chicago Comer Children's Hosp, Chicago, IL; Tod Children's Hospital-Forum Health, Youngstown, OH; Toledo Children's Hospital, Toledo, OH; Tripler Army Medical Center (USOC), Tripler AMC, HI; Tulane Univ./Tulane Univ. Hospital and Clinic, New Orleans, LA; UCLA School of Medicine, Los Angeles, CA; UCSF School of Medicine, San Francisco, CA; United States Air Force Med Ctr, Keesler AT (USOC), Keesler AFB, MS; University Medical Center Groningen, Groningen, GR, Netherlands; University of Alabama, Birmingham, AL; University of Arizona Health Sciences Center, Tucson, AZ; University of Arkansas, Little Rock, AR; University of California, Davis, Sacramento, CA; University of California, Irvine, Orange, CA; University of Florida, Gainesville, FL; University of Illinois, Chicago, IL; University of Iowa Hospitals & Clinics, Iowa City, IA; University of Kansas Medical Center, Kansas City, KS; University of Maryland at Baltimore, Baltimore, MD; University of Massachusetts Medical School, Worcester, MA; University of Medicine and Dentistry of New Jersey, New Brunswick, NJ; University of Miami School of Medicine, Miami, FL; University of Minnesota Cancer Center, Minneapolis, MN; University of Mississippi Medical Center Children's Hospital, Jackson, MS; University of Missouri at Columbia, Columbia, MO; University of Nebraska Medical Center, Omaha, NE; University of New Mexico School of Medicine, Albuquerque, NM; University of North Carolina at Chapel Hill, Chapel Hill, NC; University of Oklahoma Health Sciences Center, Oklahoma City, OK; University of Rochester Medical Center, Rochester, NY; University of South Alabama, Mobile, AL; University of Texas Health Science Center at San Antonio, San Antonio, TX; University of Texas Medical Branch, Galveston, TX; University of Vermont College of Medicine, Burlington, VT; University of Virginia Health Sciences Center, Charlottesville, VA; University of Wisconsin—Childrens Hosp Madison, Madison, WI; UT Southwestern Medical Center, Dallas, TX; Vanderbilt Children's Hospital, Nashville, TN; Via Christi Regional Medical Center, Wichita, KS; Virginia Commonwealth Univ Health System-MCV, Richmond, VA; Wake Forest University School of Medicine, Winston-Salem, NC; Walter Reed Army Medical Center (USOC), Washington, DC; Warren Clinic, Inc., Tulsa, OK; Washington University Medical Center, St. Louis, MO; Wellington Children's Hospital, Wellington, New Zealand; Wesley Medical Center, Wichita, KS; West Virginia University HSC at Charleston, Charleston, WV; West Virginia University HSC at Morgantown, Morgantown, WV; Wichita CCOP, Wichita, KS; Wilford Hall Medical Center, Lackland AFB, TX; William Beaumont Hospital, Royal Oak, MI; Winthrop University Hospital, Mineola, NY; Women's and Children's Hospital, Adelaide, North Adelaide, SA, Australia; and Yale University School of Medicine, New Haven, CT.