Purpose: Heat shock protein 90 (Hsp90) is essential for the posttranslational control of many regulators of cell growth, differentiation, and apoptosis. 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG) binds to Hsp90 and alters levels of proteins regulated by Hsp90. We conducted a phase I trial of 17-AAG in pediatric patients with recurrent or refractory neuroblastoma, Ewing's sarcoma, osteosarcoma, and desmoplastic small round cell tumor to determine the maximum tolerated dose, define toxicity and pharmacokinetic profiles, and generate data about molecular target modulation.

Experimental Design: Escalating doses of 17-AAG were administered i.v. over 1 to 2 h twice weekly for 2 weeks every 21 days until patients experienced disease progression or toxicity. harmacokinetic and pharmacodynamic studies were done during cycle 1.

Results: Fifteen patients were enrolled onto dose levels between 150 and 360 mg/m2; 13 patients were evaluable for toxicity. The maximum tolerated dose was 270 mg/m2. DLTs were grade 3 transaminitis and hypoxia. Two patients with osteosarcoma and bulky pulmonary metastases died during cycle 1 and were not evaluable for toxicity. No objective responses were observed. 17-AAG pharmacokinetics in pediatric patients were linear; clearance and half-life were 21.6 ± 6.21 (mean ± SD) L/h/m2 and 2.6 ± 0.95 h, respectively. Posttherapy increases in levels of the inducible isoform of Hsp70, a marker of target modulation, were detected in peripheral blood mononuclear cells at all dose levels.

Conclusion: 17-AAG was well tolerated at a dose of 270 mg/m2 administered twice weekly for 2 of 3 weeks. Caution should be used in treatment of patients with bulky pulmonary disease.

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an essential role in stress tolerance; protein folding; and control of many regulators of cell growth, differentiation, and apoptosis (1). 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG; NSC 330507) binds the atypical NH2-terminal ATPase of Hsp90 and prevents the chaperone from cycling between its ADP- and ATP-bound conformations. This drug-induced alteration of Hsp90 function in turn stimulates proteasome-mediated degradation of a number of cancer-associated molecules. These include ErbB2, RAF-1, steroid hormone receptors, the type I insulin-like growth factor receptor, mutant p53, telomerase, AKT, FLT3, cyclin-dependent kinase 4, CHK1, BCR-ABL, and trkB (215). Antitumor activity of Hsp90 inhibitors has been reported in experimental models of neuroblastoma, Ewing's sarcoma, and osteosarcoma (6, 1618).

Several phase I studies of 17-AAG have been done in adults (1923). Toxicities have included transaminitis, hyperbilirubinemia, anemia, thrombocytopenia, pancreatitis, fatigue, diarrhea, and nausea (1922). This study was conducted to determine the maximum tolerated dose of 17-AAG in pediatric patients, to define its toxicity and pharmacokinetic profiles, and to evaluate the extent to which 17-AAG modulates levels of Hsp90-associated proteins in peripheral blood mononuclear cells (PBMC).

Patients with histologic diagnoses of neuroblastoma, Ewing's sarcoma, osteosarcoma, desmoplastic small round cell tumor, or rhabdomyosarcoma that progressed despite standard therapy or for which no standard therapy was available were eligible. Patients were required to have life expectancies >8 weeks and Karnofsky scores ≥70% if >10 years of age, or Lansky scores ≥70% if ≤10 years of age. Patients must have been under 21 years of age at diagnosis. Other requirements were recovery from acute toxic effects of prior therapies; negative pregnancy test for women of child-bearing potential; adequate organ function as defined by absolute neutrophil count ≥750/mm3, platelet count ≥75,000/mm3, serum creatinine ≤1.5 × upper limit of normal for age or glomerular filtration rate ≥70 mL/min/1.73 m2, transaminases ≤2.5 × institutional upper limit of normal, bilirubin ≤1.5 mg/dL, albumin ≥2 g/dL, international normalized ratio ≤1.5, and shortening fraction ≥28% by echocardiogram or multigated acquisition scan. Patients with central nervous system metastases, uncontrolled illnesses, or egg allergies were not eligible. Chemotherapy and localized radiotherapy were not permitted within 4 weeks of study entry, and ≥6 months had to have elapsed since irradiation of ≥50% of the pelvis or other substantial bone marrow irradiation. Treatment with biological agents (including monoclonal antibodies) was not permitted within 14 days of study entry, and treatment with retinoids or growth factors was not permitted within 7 days. Patients who had undergone stem cell transplantation were eligible if ≥3 months had elapsed since autologous transplantation or if ≥6 months had elapsed since allogeneic transplantation, provided that there was no evidence of active graft-versus-host disease. Administration of agents that alter CYP3A4 activity was not permitted. Corticosteroids were permitted only for treatment of adrenal crisis or allergic reactions. Due to adverse events (QTc prolongation and/or atrioventricular conduction abnormalities) reported in adults treated with Hsp90 inhibitors, eligibility criteria were amended to include normal serum potassium, magnesium, and ionized calcium, as well as documentation of normal QT intervals. Patients with heart failure, history of significant arrhythmias, or prior chest radiation were excluded. The study was approved by institutional review boards at Pediatric Oncology Experimental Therapeutics Investigators' Consortium institutions; written informed consent was obtained from patients/legal guardian(s).

Drug administration. 17-AAG (NSC 330507) and egg phospholipid diluent (NSC 704057) were supplied by the National Cancer Institute (Bethesda, MD). 17-AAG was supplied in glass vials containing 50 mg of 17-AAG in 2.0-mL DMSO. Egg phospholipid diluent as supplied in 50-mL vials containing 48 mL of 2% egg phospholipids and 5% dextrose in water for injection. Diluent was added to drug to give a final concentration of 1 mg/mL. Diluted drug was dispensed in glass bottles; infusions were completed within 8 h of preparation. Most infusions were given over 1 h; however, drug could be administered over up to 2 h. Patients received 17-AAG twice weekly for 2 weeks; cycles were repeated every 3 weeks. Three to six patients (including at least one patient <13 years of age) were enrolled per dose level. If none of the first three patients at a given dose level experienced dose-limiting toxicity (DLT), three additional patients were treated at the next dose level. If one of three patients experienced DLT, up to three more patients were treated at the same dose level. If an additional patient experienced DLT, subsequent patients were treated at the next lower dose level, provided fewer than six patients had been treated at that level previously. The maximum tolerated dose was defined as the highest dose level wherein less than one third of patients (in a cohort of more than three patients) experienced DLT. The starting dose was 150 mg/m2; subsequent dose levels were 200, 270, and 360 mg/m2. Intrapatient dose escalation was not permitted.

Toxicity. Toxicities were graded according to National Cancer Institute Common Toxicity Criteria version 3.0. DLT was defined as any nonhematologic, drug-related toxicity grade ≥3 occurring in the first cycle of therapy, with the exception of fatigue/asthenia, transient arthralgia/myalgia, and alopecia. Grade 3 or 4 nausea, vomiting, and diarrhea were considered dose limiting if optimal medical therapy had been administered. Grade 4 thrombocytopenia, neutropenia, or febrile neutropenia was considered dose limiting in patients without progressive marrow disease. Patients with DLT could be re-treated at doses reduced by one dose level for a maximum of two dose level reductions. Patients who experienced toxicity that did not constitute DLT could be re-treated on resolution of toxicity to grade ≤1 or return to baseline. Treatment could be delayed for no longer than 3 weeks.

Pretreatment and follow-up studies. At the start of each cycle, complete histories and physical examinations were done. Complete blood counts, electrolytes (including magnesium, phosphorous, and calcium), transaminases, bilirubin, blood urea nitrogen, creatinine, and prothrombin time were obtained at baseline, weekly during the first cycle, before each subsequent cycle, and as clinically indicated. For patients enrolled after the study was amended, electrocardiograms were done within 24 h before and after drug administration during each cycle. Pregnancy tests for women of childbearing potential and urinalyses were obtained ≤7 days before initiation of treatment. Disease measurements were done within 4 weeks before start of treatment and every 6 weeks thereafter. Responses in target lesions were assessed according to Response Evaluation Criteria in Solid Tumors criteria (24). Positron emission tomography with [18F]fluorodeoxyglucose was done on an optional basis.

Pharmacokinetic studies. Serial blood samples (2 mL) were collected in heparinized tubes before the first dose of 17-AAG and at 0.5, 0.92, 1.25, 1.5, 2, 4, 8, and 24 h after the start of the infusion. Samples were centrifuged at 1,000 × g for 10 min and plasma was stored at −70°C until analysis. Concentrations of 17-AAG and its active metabolite 17-AG were measured using a previously described, validated high-performance liquid chromatography assay (25). The lower limit of quantitation of the assay was 0.1 μmol/L for both 17-AAG and 17-AG, and the assay was linear between 0.1 and 25.6 μmol/L. The time cycles of 17-AAG and 17-AG in plasma were analyzed noncompartmentally using the LaGrange function (26), as implemented by the computer program LAGRAN (27). Total body clearance for 17-AAG was calculated as dose per area under the curve (AUC).

Molecular targets analyses. To assess potential effects of 17-AAG exposure on Hsp90 function, blood was obtained before and 24 h after drug therapy. Volume drawn at each time point was calculated using the following formula: volume (mL) = weight (kg) × 0.65. Blood was placed into EDTA-containing tubes and kept on ice. Gradient centrifugation was done within 1 h of blood collection using Oncoquick (Grenier Bio-One, Longwood, FL) separation tubes (28). Cell pellets were frozen at −70°C until shipped for further analyses. Pellets were thawed and cells lysed in CHAPS buffer (first 10 patients) or TNES buffer [50 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 2 mmol/L EDTA, 1% NP40] with 1 mmol/L phenylmethylsulfonyl fluoride, 20 μg/mL leupeptin, and 20 μg/mL aprotinin (subsequent patients). Lysates were clarified by centrifugation at 14,000 × g for 20 min at 4 °C; protein concentrations were determined with bicinchoninic acid reagent (Pierce, Rockford, IL). Proteins were denatured in reducing loading buffer by heating samples to 95°C for 5 min. Equal amounts of total protein were size fractionated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary antibodies including anti-Hsp72 (C-92F3A-5, Stressgen, Victoria, British Columbia, Canada), anti-AKT (not phosphospecific; Cell Signaling Technologies, Beverly, MA), anti–type I insulin-like growth factor receptor (C-20, Santa Cruz Biotechnology, Santa Cruz, CA), and anti–β-actin (Sigma-Aldrich, St. Louis, MO) as previously described (6). Peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA), chemiluminescent substrate (SuperSignal West Pico, Pierce), and exposure to Kodak XAR-5 film were used for detection.

Fifteen patients (median age, 14 years; range, 4-21 years) with neuroblastoma (6), osteosarcoma (6), Ewing's family tumors (2), and desmoplastic small round cell tumor (1) were treated with 17-AAG at dose levels between 150 and 360 mg/m2. Clinical characteristics are shown (Table 1). Patients were heavily pretreated; 80% had received ≥4 prior regimens. A total of 24 cycles (median, 2 cycles per patient) were administered. Thirteen patients were evaluable for toxicity. An 11-year-old with osteosarcoma and lung metastases experienced rapid disease progression and death during cycle 1 and was not fully evaluable. A 20-year-old with osteosarcoma and bulky pulmonary metastases who required supplemental oxygen and had a moderate metabolic acidosis before treatment was also not fully evaluable. This patient experienced intractable vomiting and diarrhea within 24 h of the first drug infusion, developed a profound acidosis, and experienced an aspiration event. He expired due to respiratory failure <48 h after initial treatment. There were no objective responses to 17-AAG therapy based on computer-assisted tomography/magnetic resonance imaging. Six patients underwent positron emission tomography imaging; results were consistent with disease progression.

Table 1.

Patient characteristics

n (%)
Age (y)  
    Median 14 
    Range 4-21 
Sex  
    Male 11 (73) 
    Female 4 (27) 
Tumor type  
    Neuroblastoma 6 (40) 
    Osteosarcoma 6 (40) 
    Ewing's sarcoma 2 (13) 
    Desmoplastic small round cell tumor 1 (7) 
Performance status (%)  
    100 9 (60) 
    90 3 (20) 
    80 2 (13) 
    70 1 (7) 
Prior therapy  
    ≥4 regimens 12 (80) 
    <4 but ≥2 regimens 2 (13) 
    <2 regimens 1 (7) 
    Previous stem cell transplant 4 (27) 
Bone marrow metastases documented before treatment 4 (27) 
n (%)
Age (y)  
    Median 14 
    Range 4-21 
Sex  
    Male 11 (73) 
    Female 4 (27) 
Tumor type  
    Neuroblastoma 6 (40) 
    Osteosarcoma 6 (40) 
    Ewing's sarcoma 2 (13) 
    Desmoplastic small round cell tumor 1 (7) 
Performance status (%)  
    100 9 (60) 
    90 3 (20) 
    80 2 (13) 
    70 1 (7) 
Prior therapy  
    ≥4 regimens 12 (80) 
    <4 but ≥2 regimens 2 (13) 
    <2 regimens 1 (7) 
    Previous stem cell transplant 4 (27) 
Bone marrow metastases documented before treatment 4 (27) 

Toxicity. DLT was observed in two of three patients treated at the 360 mg/m2 dose level (Table 2). A 5-year-old with neuroblastoma developed grade 3 hypoxia without respiratory distress following the third drug infusion. Chest radiograph, electrocardiogram, echocardiogram, blood cultures, chemistries, methemoglobin screen, and blood counts were normal. There was no evidence of progressive disease or cardiac arrhythmia. An 18-year-old with osteosarcoma treated at this dose level experienced grade 3 elevation in aspartate aminotransferase without other evidence of liver toxicity 24 h after administration of the second 17-AAG dose during cycle 2. The patient concurrently experienced grade 3 drug-related vomiting and diarrhea. Although the transaminitis occurred during the second cycle of therapy, it was considered dose limiting because of its severity and because of the liver toxicity observed in adults treated with 17-AAG. Both patients were re-treated with 17-AAG at the next lower dose level (270 mg/m2); neither experienced significant toxicity.

Table 2.

Drug dosing

Dose level17-AAG (mg/m2)Number enrolled (N = 15)Number assessable for toxicityNumber with DLTDLT
150  
200  
270  
360 Grade 3 hypoxia, grade 3 AST 
Dose level17-AAG (mg/m2)Number enrolled (N = 15)Number assessable for toxicityNumber with DLTDLT
150  
200  
270  
360 Grade 3 hypoxia, grade 3 AST 

Abbreviation: AST, aspartate aminotransferase.

Non–dose-limiting toxicities observed in other patients (Table 3) included grades 1 to 3 anemia, grade 3 neutropenia with and without fever, grades 1 and 2 thrombocytopenia, and grades 1 and 2 leukopenia. Nonhematologic toxicities during cycle 1 that were not dose limiting were gastrointestinal (grades 1 and 2 nausea and vomiting), hepatic (grades 1 and 2 elevations in transaminases or bilirubin), and neurologic (grades 1 and 2 confusion and peripheral neuropathy).

Table 3.

Drug-related toxicity

ToxicityCycle 1
Cycle 2
Grade 1Grade 2Grade 3Grade 4Grade 1Grade 2Grade 3Grade 4
Hematologic         
    Neutropenia 
    Thrombocytopenia 
    Anemia 
Respiratory (hypoxia, allergy/paroxysmal coughing) 
Hepatic (AST, ALT, albumin, bilirubin, alk. phos) 13 
Cardiovascular (bradycardia, tachycardia) 
Renal (creatinine) 
Neurologic (confusion, peripheral neuropathy) 
Gastrointestinal (anorexia, nausea, vomiting, diarrhea) 
ToxicityCycle 1
Cycle 2
Grade 1Grade 2Grade 3Grade 4Grade 1Grade 2Grade 3Grade 4
Hematologic         
    Neutropenia 
    Thrombocytopenia 
    Anemia 
Respiratory (hypoxia, allergy/paroxysmal coughing) 
Hepatic (AST, ALT, albumin, bilirubin, alk. phos) 13 
Cardiovascular (bradycardia, tachycardia) 
Renal (creatinine) 
Neurologic (confusion, peripheral neuropathy) 
Gastrointestinal (anorexia, nausea, vomiting, diarrhea) 

Abbreviations: ALT, alanine aminotransferase; alk. phos, alkaline phosphatase.

Pharmacokinetics. Pharmacokinetic sampling was done after the first dose of 17-AAG. 17-AAG pharmacokinetics were linear across the doses administered (Fig. 1). 17-AAG AUC increased from 12.2 ± 4.1 μmol/L × h at 150 mg/m2 to 39.1 ± 13.2 μmol/L × h at 360 mg/m2. 17-AAG clearance and t1/2 were 21.6 ± 6.2 l/h/m2 and 2.6 ± 0.95 h, respectively, and did not vary systematically across doses. Although 17-AG maximum plasma concentration (Cmax) and AUC also increased with 17-AAG dose, the relationships were less clear than for 17-AAG Cmax and AUC. 17-AG Cmax increased from 3.2 ± 2.2 μmol/L at 150 mg/m2 to 6.2 ± 4.6 μmol/L at 360 mg/m2, and 17-AG AUC increased from 15.7 ± 12.2 μmol/L × h at the former dose to 35.5 ± 33 μmol/L × hours at the latter. 17-AG Cmax occurred at 1.5 ± 0.4 h, and 17-AG t1/2 was 6.1 ± 4.7 h. Patients experienced extensive exposure to both 17-AAG and 17-AG, and the relative exposure to metabolite versus parent compound, as reflected by the ratio of their respective AUCs, was 1.0 ± 0.7.

Fig. 1.

Relationship between 17-AAG dosage, Cmax, and AUC. Symbols represent individual patients.

Fig. 1.

Relationship between 17-AAG dosage, Cmax, and AUC. Symbols represent individual patients.

Close modal

Molecular marker studies. Consent for sampling for molecular end point modulation studies was obtained for all subjects. Cell pellets were obtained before and after therapy from all but one patient. Variable RBC contamination or low protein content precluded the use of eight sample pairs. Adequate immunoblot data were generated from five sample pairs. Because increased levels of the inducible isoform of Hsp70 (Hsp72) indicate induction of the cellular stress response following Hsp90 inhibition, levels of Hsp72 were evaluated. Increases in band intensities corresponding to Hsp72 were observed in samples from patients treated at all dose levels (Fig. 2), indicating inhibition of Hsp90 activity. Drug-induced changes in other markers, including type I insulin-like growth factor receptor and AKT, were more variable (Fig. 2).

Fig. 2.

Immunoblot analysis of protein levels in patient PBMCs. Blood samples were obtained before and 24 h after a single dose of 17-AAG. PBMCs were isolated by gradient centrifugation. Cells were lysed and levels of type I insulin-like growth factor receptor (IGF1R), AKT, and Hsp72 in equal amounts of total protein were evaluated by immunoblotting. Actin levels were assessed as a loading control. Representative blots from individual patients treated at each dose level.

Fig. 2.

Immunoblot analysis of protein levels in patient PBMCs. Blood samples were obtained before and 24 h after a single dose of 17-AAG. PBMCs were isolated by gradient centrifugation. Cells were lysed and levels of type I insulin-like growth factor receptor (IGF1R), AKT, and Hsp72 in equal amounts of total protein were evaluated by immunoblotting. Actin levels were assessed as a loading control. Representative blots from individual patients treated at each dose level.

Close modal

17-AAG is the first inhibitor of Hsp90 function to enter clinical trials. Hsp90 is a critical component of multiprotein complexes that regulate the activity and turnover of a range of signal transduction proteins (1). More than 50 cancer-related proteins have been shown to associate with Hsp90 as clients (29). Given the redundancy and complexity of signaling pathway abnormalities in cancer cells, the ability of Hsp90 inhibitors to alter the function of multiple signaling molecules may be advantageous (30). Results from adult phase I studies indicate that 17-AAG can be administered without excessive toxicity (1922).

Several dosing schedules have been evaluated in adult trials of 17-AAG, including daily × 5 every 3 weeks (19); twice weekly for 2 of 3 and 3 of 4 weeks (23); and weekly (2022). Evaluation of serial PBMC samples from patients treated weekly showed that biological effects could be observed for 3 to 4 days following drug administration (22), and twice-weekly drug dosing was shown to be safe in adults (23). Therefore, a twice-weekly schedule was chosen for this study. Because the use of a restricted panel of antibodies for analysis of circulating tumor cells was initially planned, diagnoses were limited to neuroblastoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, and desmoplastic small round cell tumor.

Although subjects were heavily pretreated, toxicities observed were similar to those seen in adults, and the maximum tolerated dose of 270 mg/m2 identified for pediatric patients exceeds the adult maximum tolerated dose of 200 mg/m2 (23). Although four patients had previously undergone stem cell transplantation and four had marrow disease, hematologic toxicities were mild to moderate and were not dose limiting. Elevation in liver enzymes was dose limiting in this study, as it was in several adult studies of 17-AAG (19, 20, 31). Transient hypoxemia temporally related to 17-AAG administration was dose limiting in one patient who did not have underlying lung disease. Two additional patients experienced hypoxemia shortly after drug administration. One had radiographic evidence of progressive pulmonary disease; hypoxemia was attributed to the underlying disease. The other patient had hypoxemia at baseline due to bulky pulmonary metastases and subsequently developed respiratory failure as a terminal event. This patient also had diarrhea and emesis, which occurred in the context of a preexisting metabolic acidosis. In a study of 17-AAG in adults, two patients were reported to have experienced hypoxia related to treatment (20). One of these patients had preexisting pulmonary disease and died shortly after the first drug infusion, as did the two patients who died during our study. Although there were eight pediatric patients with bulky pulmonary disease who had no respiratory symptomatology during 17-AAG therapy, changes in pulmonary physiology in patients with extensive chest disease could potentially predispose to respiratory compromise, either related to 17-AAG or to its DMSO/phospholipid diluent.

Excellent compliance was achieved in submission of specimens for correlative studies. Complete sets of samples for pharmacokinetic evaluation were received for 14 of 15 patients. Because the molecular end point being studied was the change in the level of the inducible isoform of Hsp70, care was taken to avoid ex vivo induction of the cellular stress response during sample transport. For this reason, on-site isolation of PBMCs was required, and it was feasible to collect and process PBMCs from children treated at multiple sites. Furthermore, it was possible to generate data about molecular target modulation from samples from smaller children, as indicated by the immunoblot results from samples from an 18-kg 4-year-old (Fig. 2C).

For molecular target modulation studies, mononuclear cells were isolated with Oncoquick density centrifugation columns, which were designed to enrich for circulating tumor cells (28). However, we found that cell pellets contained predominantly lymphocytes. Consequently, the drug-induced modulation of end points shown in Fig. 2 largely reflects the response of normal somatic tissue rather than tumor. Effects of Hsp90 inhibitors are likely to be different in tumor cells compared with normal cells due to alterations in protein homeostasis and Hsp90 utilization in advanced malignancies (32, 33). However, federal regulations prohibit serial tumor biopsies in children unless biopsies are likely to benefit the individual child involved. Therefore, although depletion of cancer-related Hsp90 client proteins has been shown in tumor tissues in adults treated with 17-AAG (22), we could perform target modulation studies only in PBMCs. Furthermore, as expected in the phase I setting, patients with different diseases were treated at different dose levels. Study of a cohort of uniformly treated patients is needed before relationships between modulation of particular molecular end points and antitumor effect can be assessed. Because patterns of changes of greatest importance for cell survival and proliferation are likely to vary among different types of pediatric solid tumors, tumor-specific studies are also needed.

In conclusion, a 17-AAG dose of 270 mg/m2 can be safely administered twice weekly for 2 of 3 weeks to heavily pretreated pediatric patients with recurrent and refractory solid tumors; however, 17-AAG should be used with caution in patients with bulky pulmonary disease. The pharmacokinetics of 17-AAG and its active metabolite 17-AG in children are similar to those observed in adults (21), and evidence of biological activity in PBMCs has been documented. Pediatric studies of this agent in combination with conventional chemotherapy and in combination with other molecularly targeted drugs are planned.

Grant support: 1 UO1-6985604 Fund #0203; Science Applications International Corporation-Frederick, Inc., contract no. 25XS011, U54CA090821; the Caitlin Robb Foundation; Raise a Racquet for Kids; the PANDA Foundation; and National Center for Research Resources, NIH grants M01 RR-00095, M01 RR-00069, and M01 RR-00082.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. David Spriggs for programmatic support, Karima Yataghene and Elizabeth DeKosko for study coordination and data oversight, and Jessica Venable for technical assistance.

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