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Departments of Pediatrics (A.P., K.M.P., K.J.U., D.M.B., L.L.R., K.S.B.), Genetics, Cell Biology and Development (A.P.), Biostatistics (T.L.B.), Medicine (S.K.R.), and Laboratory Medicine and Pathology (D.M.B.), University of Minnesota, Minneapolis, Minnesota 55455
Address all correspondence and requests for reprints to: Anna Petryk, Department of Pediatrics, University of Minnesota, 13-124 PWB, MMC 404, 516 Delaware Street SE, Minneapolis, Minnesota 55455. E-mail: petry005{at}umn.edu.
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Abstract |
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Objective: The objective of the study was to determine the incidence, timing, magnitude, and possible predictors of bone loss in children after HCT.
Patients and Design: The study population included 49 patients (age 5–18 yr) who were eligible to receive HCT at the University of Minnesota. The patients were evaluated at baseline, 100 d, 6 months, and 1 yr after HCT. Lumbar BMD (LBMD) was assessed by dual-energy x-ray absorptiometry.
Results: The number of patients with osteopenia increased from 18% at baseline to 33% 1 yr after HCT, and with osteoporosis from 16–19%. Mean areal LBMD z-score decreased from –0.56 to –1.1 by 6 months (n = 27) and at 1 yr was –0.94 (n = 21), which was significant compared with standard normal distribution (P = 0.004 and P = 0.022, respectively). The absolute loss of bone mineral corresponded to a 5.3% reduction in areal LBMD and a 4.8% reduction in volumetric LBMD. The level of bone-specific alkaline phosphatase decreased by 30% by d 100 (P = 0.009), followed by recovery toward baseline by 6 months. The level of osteocalcin greater than 6.5 ng/ml at d 100 predicted recovery from the initial bone loss by 1 yr. A reduction in LBMD at 6 months correlated with a cumulative dose of glucocorticoids.
Conclusion: This study demonstrates that bone loss is common in children after HCT and is primarily due to suppression of bone formation. Further studies are necessary to validate osteocalcin as a predictive biomarker.
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Introduction |
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Studies in adult survivors of childhood cancer demonstrate that BMD is reduced at multiple skeletal sites and thus support the premise that inadequate BMD accrual during childhood may have long-term adverse effect on BMD in adulthood (6, 7, 8, 9). The etiology of bone loss in patients undergoing treatment for malignancy is multifactorial and includes the negative effects of the chemotherapeutic agents, glucocorticoids, radiation, hypogonadism, abnormal vitamin D metabolism, reduced physical activity, graft vs. host disease (GVHD), and the malignant process per se (2, 6, 10, 11, 12).
HCT has been implicated as an additional causative factor in bone loss, frequently compounding the negative effects of prior treatment of malignancy on bone metabolism (1, 2, 13, 14, 15, 16, 17). In adults, significant bone loss occurs during the first 6 months after transplantation and is thought to be due to a combination of reduced bone formation and increased bone resorption (1, 15). The prevalence of osteopenia in adults after HCT is 39–53% and the prevalence of osteoporosis is 7–20% (13, 16, 18). To our knowledge, no prospective studies of BMD in a pediatric post-HCT population have been reported, but cross-sectional studies show that BMD is reduced in children after transplantation with mean BMD z-scores between –0.5 and –0.9, and as low as –4 (19, 20, 21, 22). In a recent retrospective analysis conducted at a median of 5 yr after HCT, 26% of children had osteopenia and 21% had osteoporosis based on quantitative computed tomography (22). The goals of this prospective study were to determine the incidence, timing, magnitude, and potential predictors of bone loss in children after HCT and to assess the changes in bone metabolism.
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Subjects and Methods |
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From January 2002 to January 2004 consecutive patients between 5 and 18 yr of age evaluated to undergo HCT for a blood disorder, cancer, metabolic disorder, or immune deficiency at the University of Minnesota were offered participation in the study. Of 77 eligible patients, 49 were prospectively enrolled (64%, 20 females and 29 males). Diagnoses included: Fanconi anemia (FA) (n = 12), acute lymphoblastic leukemia (ALL) (n = 10), acute myeloid leukemia (AML) (n = 8), adrenoleukodystrophy (n = 6), aplastic anemia (AA) (n = 3), chronic myeloid leukemia (n = 3), metachromatic leukodystrophy (n = 3), non-Hodgkin lymphoma (n = 1), combined variable immune deficiency (n = 1), X-linked lymphoproliferative disorder (n = 1), and Ewing’s sarcoma (n = 1). The mean chronological age of the patients at the time of HCT was 10.2 ± 3.7 yr (range 5.1–17.9), bone age 10.1 ± 3.9; 12 females and 18 males were prepubertal at the time of enrollment. The mean height SD score was –0.34 ± 1.61 (mean ± SD, n = 49).
Forty-seven patients received allogeneic HCT and two received autologous HCT. Preparative regimens consisted of cyclophosphamide, fludarabine, and 450 cGy single fraction total body irradiation (TBI) for all FA patients except one, who received only fludarabine and cyclophosphamide; cyclophosphamide and 600 cGy fractionated TBI for AA; cyclophosphamide and 1320 cGy fractionated TBI (1350 cGy for two patients with ALL) for ALL, AML, non-Hodgkin lymphoma; busulfan, cyclophosphamide for metabolic storage disorders (and one patient with AML); and fludarabine, melphalan for immune deficiencies. All patients received cyclosporine in combination with either methotrexate, mycophenolate, or methylprednisolone for GVHD prophylaxis. Total steroid exposure (expressed as prednisone equivalent in milligrams) included any steroid received with the HCT preparative regimen and for GVHD prophylaxis or treatment.
The patients were evaluated before HCT and 100 d, 6 months, and 1 yr after HCT. The patients were examined for Tanner stage of pubertal development and growth parameters by a pediatric endocrinologist at all time points (23, 24). The primary endpoints of the study were the 6- and 12-month changes in lumbar BMD (LBMD) by dual-energy x-ray absorptiometry (DXA). Changes in the markers of bone turnover were considered secondary endpoints. Of 49 patients enrolled in the study, 19 had DXA study at all three time points and an additional two patients had DXA study only at baseline and at 1 yr. Of 28 patients who did not return for a 1-yr follow-up DXA scan, 15 patients died, three patients had a relapse, and 10 patients were lost to follow up.
The study was approved by the Institutional Review Board at the University of Minnesota. Informed consent was obtained before enrollment.
Bone densitometry
Areal BMD (grams per square centimeter) of the lumbar spine (LBMDA) was measured in L2–L4 region by DXA using a Lunar Prodigy (DXA, software version 5.0; General Electric, Madison, WI) at baseline, 6 months, and 12 months after HCT. Pediatric software was used for children less than 30 kg. The results of the scans are expressed as age-normalized z-scores (SD from the mean for age- and sex-matched controls) (25). Quality assurance was performed daily on the densitometer, and monthly phantom spine scans were performed to assess precision of measurements. The coefficient of variation (CV) for repeated measures of the phantom spine is 0.136. Volumetric LBMD (LBMDV) (grams per cubic centimeter) was calculated based on the LBMDA and the width of the lumbar vertebrae according to the following formula: LBMDA x [4/(
x width] (25).
Osteopenia was defined as a z-score between 1 and 2 SD below the mean, and osteoporosis was defined as a z-score more than 2 SD below the mean (22, 26). This classification was chosen for quantitative illustration of bone loss and for simplicity of nomenclature.
Laboratory tests and bone age
Serum levels of calcium, phosphorus, and magnesium were measured by standard methods. Bone formation was assessed by measuring the levels of bone-specific alkaline phosphatase (BSAP) and osteocalcin (OCN) (markers of early and mature osteoblast function) in the serum (27). Urinary N-telopeptide was used as a marker of bone resorption.
BSAP and OCN were obtained in the early morning hours. BSAP was measured by a chemiluminescent immunoassay (DXi800; ARUP Laboratories, Salt Lake City, UT). Serum human OCN was measured by RIA (Diasorin, Inc., Stillwater, MN) between March 2002 and December 2003, and by chemiluminescence on the DPC Immulite analyzer (Diagnostic Products Corp., Los Angeles, CA) after January 2004 (due to discontinuation of the previous methodology). The sensitivity of RIA was 0.2 ng/ml, the inter and intraassay CVs were less than or equal to 11.2% at a mean of 4.8 ng/ml and less than or equal to 7.0% at a mean of 1.25 ng/ml, respectively. The sensitivity of chemiluminescence was 0.1 ng/ml, and the inter and intraassay CVs were less than or equal to 5.6% at a mean of 6.3 ng/ml and less than or equal to 2.3% at a mean of 4.4 ng/ml, respectively. The correlation between RIA and chemiluminescent assay was 0.886 as determined by comparison of 53 samples. The values obtained by chemiluminescent assay were converted using linear correlation between the two assays.
Collagen cross-linked N-telopeptide was measured in second void morning urine by ELISA/EIA (Osteomark NTx; Wampole Laboratories, Inc., Princeton, NJ). The sensitivity of the assay was 20 nM bone collagen equivalents (BCE), and the inter and intraassay CVs were less than or equal to 6.0% at a mean of 362 nM BCE and less than or equal to 19% at a mean of 26 nM BCE, respectively.
Sex hormone levels were measured in females older than 10 yr and males older than 11 yr. Ultrasensitive estradiol was assessed by RIA (Diagnostic Systems Laboratories, Inc., Webster, TX). Testosterone, LH, and FSH levels were measured by a chemiluminescent immunoassay (Bayer Advia Centaur, Tarrytown, NY).
Bone age was assessed based on the x-rays of the left hand and wrist using the method of Greulich and Pyle (28).
Dietary questionnaire
Dietary intake was assessed at baseline and 1 yr after HCT by a food frequency questionnaire designed for children (Block Kids 2000; Block Dietary Data Systems, Berkeley, CA). Analysis was performed using the Dietary Analysis System (version 4.01, National Cancer Institute, 1997).
Statistical methods
To analyze the association between one continuous measure and a continuous outcome, e.g. bone loss, linear regression analysis was used. This was simplified to a t test when comparing a continuous outcome between two groups. To compare the distribution of standardized BMD values to a normal population, we compared the collected data to simulated datasets from a standard normal distribution (25). Simulations were run 1000 times. Regression output was calculated to adjust L2–L4 z-scores for bone age.
To examine the pattern of BMD over time, a linear mixed effects model was used (29). Time is fit as a random effect because outcomes are repeated measures over time, and any other important covariates are fixed effects. This model was also used to look at biomarkers over time, such as OCN, N-telopeptide, or BSAP. A Student t test was used to compare steroid exposure between progression and recovery groups.
To examine the absolute change in BMD over a 1-yr period, the observed changes in LBMD were compared with the expected increase within age and gender groups. A random effects model was fit to test whether the decrease over time was statistically significant. This mixed effects model fit time as a random effect and bone age as a continuous fixed effect.
To explore the predictive power of OCN as a biomarker, we fit a classification and regression tree (30). This method seeks the optimal cut-off point to divide a continuous measure into binary variable by minimizing the residual deviance in the model. We used this method to determine the optimal threshold for OCN levels that would predict recovery of BMD between 6–12 months of the study.
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Results |
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The average LBMDA z-score at baseline was –0.56, which was of borderline statistical significance compared with the general population (P = 0.05). After HCT, there was a significant reduction in LBMDA (Table 1
). By 6 months, mean LBMDA z-score decreased from –0.56 to –1.1 (n = 27, P = 0.004) and at 1 yr was –0.94 (n = 21, P = 0.022). The changes in z-score over time were significantly different from the standard normal distribution. LBMDA adjusted for bone age was also reduced compared with baseline by 0.51 SD at 6 months (P = 0.002) and by 0.37 SD at 1 yr (P = 0.01) (Table 1).
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). A change in height SDS for these 19 patients was not statistically significant and did not correlate with a change in BMD z-score. This was also true for the entire study population, which argues against the possibility that poor growth alone could explain a reduction in LBMDA in these patients (31). The proportion of patients with osteopenia increased from 18.4% at baseline to 33.3% at 6 months, and the proportion of patients with osteoporosis increased from 16.3% at baseline to 25.9% at 6 months. At 1 yr, 52.3% of patients had either osteopenia (33.3%) or osteoporosis (19.0%).
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). The differences were statistically significant for both LBMDA (Fig. 1A) and LBMDV (Fig. 1B) (P < 0.0001). The differences between expected and observed LBMD resulted not only from inadequate increase in BMD, but actual loss in LBMD (Table 3). The most significant bone loss occurred by 6 months and corresponded to 5.3% reduction in LBMDA and 4.8% reduction in LBMDV.
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, A and B). Based on LBMDA z-scores at 6 months and 1 yr compared with baseline, the patients were divided into three groups: stable group, in which there was no significant decrease in LBMDA at 6 months or 1 yr; recovery group, in which there was a substantial LBMDA loss at 6 months, followed by the recovery of more than 50% of the initial loss; and progression group, in which LBMDA loss at 6 months was followed by either no recovery or the recovery of less than 50% by 1 yr. The hypothesis was that the recovery group might show different biochemical characteristics that favor recovery from the initial bone loss as opposed to the progression group.
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The trends in the levels of the markers of bone formation and resorption over time are shown in Fig. 3. Only the changes in BSAP levels were statistically significant (P = 0.009), although similar trends were seen for OCN and N-telopeptide (Fig. 3A). The level of BSAP decreased by 30% after HCT and reached a nadir by d 100, followed by a progressive return toward baseline by 6 months and significantly above baseline by 1 yr. The decreases in OCN and N-telopeptide levels during the first 100 d after HCT were not statistically significant (P = 0.15 and P = 0.17, respectively).
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). No significant differences were found in BSAP or N-telopeptide levels between the recovery and progression groups. Predictive value of OCN
The optimal value of OCN was 6.5 ng/ml at d 100. All six patients in the progression group had an OCN level less than 6.5 ng/ml (OCN level was not available for one patient at d 100); five of seven patients in the recovery group had an OCN level more than 6.5 ng/ml and two patients had OCN less than 6.5 ng/ml. Thus, a value of OCN less than 6.5 ng/ml had a 100% sensitivity and 71% specificity for the progression group. A predictive value of low OCN level was 75%.
Risk factors for bone loss
A positive relationship was detected between the cumulative dose of prednisone and the change in LBMDA z-score (R2 = 0.23, P = 0.011). The progression group was exposed to higher doses of prednisone than the recovery group (5624 ± 4657 vs. 1364 ± 1323 mg, P = 0.04). No relationship was found between radiation dose, body mass index, levels of phosphorus, magnesium, calcium, or dietary calcium, dietary vitamin D, and a change in LBMDA z-score when analyzed for all 49 patients and when compared between progression and recovery groups. Among patients who survived 1 yr after HCT, patients with ALL or AML were at higher risk of bone loss in the first 6 months than patients with FA or AA (decrease in LBMDA z-score of 0.83 compared with 0.2, P = 0.04) (Fig. 3B). Contributing factors may have been higher steroid and radiation doses, although TBI doses cannot be directly compared because of differences in the fractionation schedules (single fraction for FA and fractionated for AA and leukemia).
Endocrine function
Both growth hormone deficiency (GHD) and hypogonadism can negatively affect BMD. A change in height SDS was not statistically significant for the 19 patients who were followed longitudinally at all study points. One of 19 patients was diagnosed with GHD before HCT (Fig. 2B). Because growth hormone stimulation testing was beyond the scope of this 1-yr study, it remains to be determined how many patients developed GHD after HCT, although based on the literature, the likelihood of GHD is high (17).
One of seven females developed primary gonadal failure (FSH > 40 IU/liter), and one of 12 males developed hypogonadotropic hypogonadism (Fig. 2B).
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Discussion |
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Bone loss in adults after HCT is associated with an increased risk of fractures. Nontraumatic fractures occur in 10.6% of patients within 3 yr after HCT (16). Although the exact incidence of fractures in children after HCT is unknown, a reduction in BMD of 1 SD in children with a previous history of forearm fracture increases the risk of fractures by a factor of two, similar to the risk of fracture in adults (33, 34, 35). Because routine radiographs were not performed in our patients (except for a bone age x-ray, which detected a fracture in one of the study patients), a diagnosis of fracture would have been based on self-reporting and would likely exclude less symptomatic cases, for example vertebral compression fractures. An additional potential adverse effect of vertebral fractures in children is impaired longitudinal growth. The observation that fractures in adults post-HCT occur at higher BMD than expected suggests that BMD alone may not be the only indicator of bone health in post-HCT patients, because it does not adequately reflect altered bone architecture due to transplant itself or transplant-related treatment (16). The relationship between inadequate bone mass increment during childhood and future osteoporosis during adulthood is implied by studies of young survivors of childhood cancer, but is limited by the lack of long-term prospective studies in children. Because BMD continues to increase during childhood (25), it is conceivable that some catch-up in BMD z-scores could occur depending on the age and pubertal status.
A reduction in BMD is thought to be due to a combination of reduced bone formation and increased bone resorption (1, 15). Our data show a reduction in the markers of bone formation in children after HCT, similar to the reduction observed in adult patients (1, 14, 15), except that the decrease is more prolonged in some cases. Although, in adults, the level of OCN is above baseline at 3 months, in children, in the progression group the levels of OCN do not recover until 6 months. Unlike studies in adults, our study did not reveal an increase in bone resorption. On the contrary, we observed a trend toward a decrease in urinary N-telopeptide level after HCT, suggesting an adynamic bone state similar to that reported after renal transplantation (36). However, our evaluation was limited to the urinary levels of N-telopeptide, and did not include a serum marker, carboxyl-terminal cross-linked telopeptide of type I collagen (ICTP), which was increased throughout the observation period in adult patients (1, 14, 15). Because bone formation is dependent on the proper balance between osteoblastic and osteoclastic activity, net bone loss in the absence of increased bone resorption points to reduced bone formation due to impaired osteoblastic activity as a primary cause of reduced BMD in children after HCT.
Osteoblasts are derived from bone marrow stromal cells and are thought to be of recipient origin unlike osteoclasts, which are derived from the peripheral mononuclear cells and are of donor origin (1). Myeloablative therapy before HCT has been shown to adversely affect osteogenic differentiation (37, 38). The ex vivo studies have demonstrated that differentiation of bone marrow stromal cells into osteoblasts is impaired after HCT even for as long as 6–10 yr (1, 37). Thus, the ability to regenerate osteoblast precursors within the host bone marrow may partially determine recovery in bone mass after HCT. Our finding that OCN levels were persistently lower in the progression group than in the recovery group is consistent with this model and likely reflects the degree to which stromal microenvironment is affected by myeloablation. The effects of chemotherapy and radiotherapy may be compounded by glucocorticoid exposure as well as hypogonadism and GHD (2, 15, 16, 17). The negative effect of glucocorticoids on BMD was also shown in this study.
In conclusion, although this study is limited by a small number of subjects and the heterogenous nature of the study population, it shows that bone loss is common in the first 6 months after HCT. The lack of demonstrable increase in bone resorption, compared with the adult studies, has potential implications for the management of osteoporosis in these patients. Further research needs to be done to investigate the mechanisms of bone loss after HCT to determine the contribution of bone resorption. Although bisphosphonates have been widely used in adults with osteoporosis, the antiresorptive agents may not be appropriate in the treatment of osteoporosis in children after HCT unless bone resorption is increased (36). Although no direct relationship was found between dietary calcium and vitamin D intake, it is prudent to recommend adequate vitamin D and calcium intake, weight bearing exercise, and appropriate hormonal replacement (39). The finding that higher OCN levels at d 100 can predict subsequent recovery from the initial bone loss may help target therapy to more vulnerable patients. However, this observation is based on a limited number of patients and further studies are necessary to validate OCN as a predictive biomarker.
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Acknowledgments |
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Footnotes |
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Abbreviations: AA, Aplastic anemia; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; BCE, bone collagen equivalent; BMD, bone mineral density; BSAP, bone-specific alkaline phosphatase; CV, coefficient of variation; DXA, dual-energy x-ray absorptiometry; FA, Fanconi anemia; GHD, growth hormone deficiency; GVHD, graft vs. host disease; HCT, hematopoietic cell transplantation; ICTP, carboxyl-terminal cross-linked telopeptide of type I collagen; LBMD, lumbar BMD; LBMDA, areal LBMD; LBMDV, volumetric LBMD; OCN, osteocalcin; TBI, total body irradiation.
This work was supported by The Genentech Center for Clinical Research in Endocrinology (to A.P.), Children’s Cancer Research Fund, University of Minnesota (to A.P.), and National Institutes of Health Grants K23-CA85503 (to K.S.B.) and M01-RR00400.
Received August 29, 2005.
Accepted December 7, 2005.
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