563409
research-article2015
JPOXXX10.1177/1043454214563409Journal of Pediatric Oncology NursingTaylor et al.
Case Studies
Evaluation of Biomarkers of Oxidative Stress and Apoptosis in Patients With Severe Methotrexate Neurotoxicity: A Case Series
Journal of Pediatric Oncology Nursing 1–6 © 2015 by Association of Pediatric Hematology/Oncology Nurses Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1043454214563409 jpo.sagepub.com
Olga A. Taylor, MPH1, Marilyn J. Hockenberry, PhD, RN, PNP, FAAN2, Kathy McCarthy, BSN, RN1, Patricia Gundy, MS3, David Montgomery, PhD4, Adam Ross, BS3, Michael E. Scheurer, PhD, MPH1, and Ida M. Moore, PhD, RN, FAAN3
Abstract Central nervous system (CNS) treatment is an essential part of acute lymphocytic leukemia (ALL) therapy, and the most common CNS treatment is intrathecal (IT) and high-dose intravenous (IV) methotrexate (MTX). Treatment with MTX may cause neurotoxicity, which is often accompanied by neurologic changes, delays in treatment, and prolonged hospital stays. This article reports clinical presentations of 3 patients with severe MTX toxicity as well as levels of oxidative stress and apoptosis biomarkers in cerebrospinal fluid (CSF). Oxidative stress was measured by oxidized phosphatidylcholine (PC), oxidized phosphatidylinositol (PI), and F2 isoprostanes; apoptosis was measured by caspase 3/7 activity. Most consistent biomarker changes in all 3 cases were increases in caspase 3/7 and F2 isoprostanes prior to acute toxicity while increases in oxidized phospholipids occurred slightly later. Progressive increases in F2 isoprostanes and caspase 3/7 activity prior to and/or during acute toxicity suggests MTX induces oxidative stress and an associated increase in apoptosis. These findings support the role of oxidative stress in MTX-related neurotoxicity. Keywords neurotoxicity, methotrexate, leukemia, oxidative stress
Introduction In the United States, acute lymphocytic leukemia (ALL) is the most prevalent cancer among children and adolescents less than 15 years of age (Howlader et al., 2013). Central nervous system (CNS)-directed treatment is an essential part of ALL therapy, and the most common CNS treatment is intrathecal (IT) and high-dose intravenous (IV) methotrexate (MTX), which are administered throughout ALL therapy (Richards, Pui, Gayon, & Childhood Acute Lymphoblastic Leukemia Collaborative Group [CALLCG], 2013). MTX increases oxidative stress in several organs, including the brain, and white matter is particularly vulnerable to oxidative stress because of its high lipid content (Inder et al., 2002; Rajamani, Muthuvel, Senthilvelan, & Sheeladevi, 2006). Recent studies confirm an association between oxidative stress levels in the cerebrospinal fluid (CSF), MTX, and CNS injury (Caron et al., 2009; Hockenberry et al., 2013; Protas, Muszynska-Roslan, Holownia,
Krawczuk-Rybak, & Braszko, 2010; Stenzel et al., 2010). This article reports clinical presentations of 3 patients with severe MTX toxicity as well as CSF levels of oxidative stress and apoptosis biomarkers. Oxidative stress was measured by oxidized phosphatidylcholine (PC), oxidized phosphatidylinositol (PI), and F2 isoprostanes; apoptosis was measured by caspase 3/7 (Illingworth & Glover, 1971; Iwai et al., 2003; Porter & Janicke, 1999; Smith, Kapoor, & Felts, 1999).
1
Baylor College of Medicine Houston, TX, USA Duke School of Nursing, Durham, NC, USA 3 University of Arizona College of Nursing, Tucson, AZ, USA 4 Southern Arizona VA Health Care System, Tucson, AZ, USA 2
Corresponding Author: Olga A. Taylor, MPH, Baylor College of Medicine, 1102 Bates Street Suite 1580 Houston, TX 77030, USA. Email: [email protected]
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Setting and Sample These 3 cases were part of a larger longitudinal study evaluating oxidative stress biomarkers in pediatric ALL patients undergoing chemotherapy. Subjects were from 2 cancer centers in the southwestern United States. Each of the center’s institutional review boards approved the study. Parents or legal guardians provided informed consent for all subjects, and subjects age 7 or older provided assent. Children were eligible for the study if they were diagnosed with ALL and being treated on Children’s Oncology Group Protocols, were between 2 years 9 months and 15 years of age at the time of diagnosis, and spoke English. Children with a preexisting medical history of neurological disorders (i.e., seizures), psychiatric disorders, or a traumatic brain injury associated with an alteration of consciousness or a developmental disability such as Down Syndrome were excluded. The patients included in this case series presented with neurological changes during treatment. Children with ALL received chemotherapy in 3 phases referred to as induction, postinduction, and continuation therapy. Induction therapy (1 month) included weekly treatment with vincristine and daunomycin (for high-risk ALL), a corticosteroid and a dose of PEG-asparaginase, and 2 IT MTX treatments. Postinduction therapy (6-8 mo) involved several courses of treatment that included asparaginase, high- or intermediate-dose IV MTX (depending on ALL protocol assignment), vincristine, doxorubicin, corticosteroid, cyclophosphamide, cytarabine, and mercaptopurine. Continuation therapy (2-3 years) consisted of daily mercaptopurine and weekly oral MTX, with monthly pulses of vincristine and a corticosteroid. Throughout therapy, study participants received CNS prophylaxis with standardized doses of IT MTX based on age: 10 mg for children aged 2 to 2.99 years, 12 mg for children aged 3 to 8.99 years, and 15 mg for children aged ≥9 years. The 3 cases presented in this article had no identified leukemia blasts in their spinal fluid at ALL diagnosis and were receiving treatment in the postinduction phase of therapy when the clinical presentation of MTX toxicity occurred.
Methods for CSF Measures of Oxidative Stress CSF samples were collected when children with ALL had scheduled lumbar punctures at the time of diagnosis and subsequently for administration of IT MTX. CSF samples were placed on ice immediately after collection and centrifuged for 10 minutes at 3000 rpm to remove any cellular debris. Processed CSF was stored at –80º C. Promega CaspaseGlo luminescence assay kits were used to measure caspase 3/7 activity as instructed by the manufacturer. Each sample was measured in duplicate wells of 96 well, half-area white plates, with a sample volume of 25
µl/well. Plates were read using a Perkin-Elmer Envision Xcite multilabel plate reader with a high-sensitivity luminometer. Caspase enzyme activity was determined by comparison to a standard curve of the purified caspase enzyme (ENZO) using linear regression analysis and data expressed as Units/Lcaspase activity. Glycerophospholipids were extracted from 1 ml of CSF, separated by normal phase high performance chromatography (HPLC). Concentration was measured using peak area (defined as the area proportional to the amount of compound that is present), which is a measure of concentration. F2 isoprostanes were measured by a competitive enzyme-linked immunoassay (ELISA) kit according to instructions (Cayman Chemical, https://www.caymanchem.com). For a full description of methods for F2 isoprostanes, please see previous publication (Hockenberry et al., 2013).
Case 1 A 13-year-old female was diagnosed with high-risk pre-B ALL. Two months after diagnosis, the patient received the first course of postinduction therapy, a phase of intensive CNS treatment, consisting of intravenous cytarabine (Ara-C), oral mercaptopurine (6-MP), and IT MTX. After completing this course of therapy, the patient exhibited neurological changes, slurred speech and unsteady gait (walking to one side), at home. A day later, she could not speak and had trouble chewing. Prior to this event, the patient received a total of 75 mg of IT MTX (5 standard doses per protocol at 15 mg/dose for patients 9 years of age or older). Upon examination of the patient in the emergency room, right-sided weakness was found, which was substantially worse than the left side. Two days after the first neurological event, progression in the patient’s altered mental status and ataxia was observed. These findings are consistent with further evolution of IT MTX toxicity, and systemic chemotherapy treatment was resumed. The patient continued on high-dose IV MTX and oral MTX per protocol but received no IT MTX. Magnetic resonance imaging (MRI) was performed and revealed multiple lesions in the white matter thought to be consistent with MTX toxicity. Two biomarkers of oxidative stress, oxidized PC and F2 isoprostanes, in the CSF increased from pretreatment to period of acute toxicity (Table 1). The highest levels of oxidized PI were observed after the acute toxicity (peak area 1 023 715; Table 1). The highest levels for all oxidative stress biomarkers were found after acute toxicity (Table 1). From pretreatment, oxidized PC had a 9-fold increase, oxidized PI had a 3-fold increase, and F2 isoprostanes had a 2-fold increase (Table 1). Caspase 3/7 activity (apoptosis biomarker) increased from pretreatment (12.56 Units/L) to the neurotoxicity episode (113.5 Units/L) and then dropped after toxicity (25.61 Units/L).
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Taylor et al. Table 1. Biomarkers of Oxidative Stress and Apoptosis. Case 1 2 3
Sample Phase
Days From Diagnosis
Pretreatment Prior to toxicity Acute toxicity Event After toxicity Pretreatment Acute toxicity Event Event After toxicity After toxicity Pretreatment Prior to toxicity Acute toxicity Event After toxicity After toxicity
0 35 42 46 106 16 30 40 42 77 85 0 44 51 58 65 114
Caspase 3/7 (Units/L) 12.56 67.95 113.5 — 25.61 66.62 60.89 — — 50.85 50.6 7.6 20.19 35.89 — 59.75 41.48
F2 Isop (pg/mL) 11.092 18.616 17.556 — 24.91 9.511 10.621 — — 13.467 12.92 4.245 4.956 6.527 — 7.142 5.86
Oxidized PC (peak area)
Oxidized PI (peak area)
17 188 15 217 27 727
321 851 934 069 158 119 — 1 023 715 674 921 180 871 — — 910 859 1 196 316 252 273 108 948 420 336 — 80 689 532 779
— 152 900 38 749 26 385 — — 20 252 20 596 8581 12 781 10 171 — 14 021 6377
Abbreviations: Isop, isoprostanes; PC, oxidized phosphatidylcholine; PI, oxidized phosphatidylinositol.
Case 2
Case 3
A 14-year-old male was diagnosed with high-risk ALL. One month later, 10 days after IT MTX administration, the patient experienced acute onset of mental changes. Prior to this event, the patient had received 2 IT doses of 15 mg of MTX per protocol. The patient presented with generalized weakness associated with diaphoresis, left greater than right upper extremity weakness, with temporary resolution. While hospitalized, the patient had a subsequent episode of generalized weakness, this time with right upper extremity greater than left upper extremity weakness. Two days after the initial onset of mental status changes, the patient experienced right-sided facial droop and mechanical aphasia as well as right upper and lower extremity hemiparesis. MRI revealed lesions in the white matter, which were consistent with MTX toxicity, and chemotherapy treatment was resumed without IT MTX. The patient received Capizzi IV MTX (begins with low dose then gradually increases as tolerated), oral MTX and was restarted on IT MTX 6 months following the neurotoxicity episode. Oxidized PI and F2 isoprostanes had an overall increase from pretreatment to after toxicity (Table 1). F2 isoprostanes had a slight increase in concentration from pretreatment to period of acute MTX toxicity (9.5 pg/mL to 10.62 pg/mL). The greatest increase in oxidized PI was observed after toxicity (peak area 1 196 316; Table 1). Caspase 3/7 activity did not change significantly during the time points evaluated.
A 12-year-old female was diagnosed with intermediate risk T-cell ALL. Approximately two months later, the patient experienced acute mental confusion, right-sided weakness, and facial drooping. Prior to the onset of neurologic symptoms the patient received two 15 mg MTX doses given IT on treatment days 7 and 14. In addition to IT, the patient received 1 dose of cyclophosphamide 13 days prior to acute neurologic symptoms, 8 doses of intravenous Ara-C (last dose was 4 days prior to neurologic symptoms), and 13 daily doses of oral 6-MP. CT scan and MRI did not reveal any brain lesions. There was approximately a 2-fold increase in oxidized PI from pretreatment to after the acute toxicity event (Table 1). The greatest increase in oxidized PI was observed in the CSF sample after the neurotoxicity event (peak area 532 779; Table 1). Caspase 3/7 activity and F2 isoprostanes concentration also increased progressively from pretreatment to acute toxicity (Table 1). F2 isoprostanes increased from 4.25 pg/mL during pretreatment to 7.14 pg/mL during acute toxicity. Caspase 3/7 activity had a more dramatic increase from 7.6 Units/L pretreatment to 59.75 Units/L during acute toxicity.
Discussion This case series is the first to our knowledge to examine CSF biomarkers of oxidative stress and apoptosis in patients with severe MTX neurotoxicity. These findings
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contribute to the growing body of evidence for the association of oxidative stress and effects of childhood cancer treatment. In this report, oxidized PC, oxidized PI, and F2 isoprostanes measured oxidative stress; apoptosis was measured by caspase 3/7 (Illingworth & Glover, 1971; Iwai et al., 2003; Porter & Janicke, 1999; Smith et al., 1999). F2 isoprostanes are formed from peroxidation of arachidonic acid, a polyunsaturated free fatty acid found in high concentrations in membrane lipids (Milne et al., 2005; Roberts & Morrow, 2000). In all 3 cases, F2 isoprostanes increased progressively prior to and/or during acute toxicity episodes. Varma et al. (2003) evaluated CSF F2 isoprostane levels in healthy children and those with traumatic brain injury (TBI). Pretreatment levels for the 3 cases reported here (range, 4.2-11.1 pg/ml; Table 1) are comparable to healthy controls (5.64 ± 8.08 pg/ml) reported by Varma et al. (2003). Values after acute toxicity, particularly for Cases 1 and 2 (24.91 pg/ml and 13.4 pg/ml, respectively; Table 1) are in the range of values found in children with TBI (15-36.59 pg/ml). These data suggest that MTX toxicity induces similar damage and increases in oxidative stress levels as children with TBI. Cell membrane phospholipids, such as PC and PI, have a high concentration of polyunsaturated fatty acids and are susceptible to oxidant attack (Girotti, 1998). In a sample of children with ALL (N = 36) who did not experience acute MTX toxicity, mean oxidized PC peak area pretreatment was 14 482.06 (SEM = 1193.41) (Hockenberry et al., 2013). The highest level of oxidized PC peak area occurred on day 8 of treatment (mean = 18 497.39; SEM = 2188.82). Oxidized PC peak area was approximately 8-fold higher in Case 1, and this patient experienced the most severe toxicity. This case study supports oxidized PC as a potential biomarker of oxidative stress in acute neurological toxicity. In this same sample of 36 children, mean oxidized PI peak area was 56 7977 (SEM = 11 8328) at diagnosis (Hockenberry et al., 2013). The greatest increase in oxidized PI also occurred on day 8 (mean = 902 886; SEM = 223 164). Although oxidized PI increased in all 3 cases, the increases were only greater in Cases 1 and 2 when compared to peak areas found in children without acute MTX toxicity. Caspase 3/7 activity is an established biomarker of apoptosis. Uzan et al. (2006) examined caspase 3 in the CSF of healthy patients and those with severe head injury. No detectable concentrations of caspase 3 activity were noted in controls. Caspase 3 activity in particular has been reported to be increased in traumatic brain injury (Harter, Keel, Hentze, Leist, & Ertel, 2001; Uzan et al., 2006). In Cases 1 and 3, CSF caspase 3/7 activity was elevated prior to (67.95 Units/L and 20.19 Units/L, respectively) and during acute neurotoxicity (113.5 Units/L to 35.89 Units/L, respectively). Of note, the
Table 2. Biomarkers in Acute Lymphocytic Leukemia (ALL) Patients Undergoing Treatment Without Evidence of Neurotoxicity. Therapy Phase Post induction
Caspase 3/7 Units/L (SEM)
F2 Isop pg/mL (SEM)
48.183 (1.99)
6.26 (0.34)
Oxidized Oxidized PI PC Peak Peak Area Area (SEM) (SEM) 14430 (500)
685 160 (96880)
Abbreviations: Isop, isoprostanes; PC, oxidized phosphatidylcholine; PI, oxidized phosphatidylinositol.
greatest increase was observed in Case 1 during the neurotoxicity event (113.5 Units/L). Although no dramatic changes were observed in Case 2, the concentration of caspase 3/7 activity was higher (range, 50.60-66.62 Units/L) than published controls and similar to that observed during acute toxicity in the other 2 cases. These patterns suggest increases in caspase 3/7 may precede increased oxidation of membrane phospholipids, perhaps due to its role in apoptosis activation. The most consistent biomarker changes in all 3 cases were increases in caspase 3/7 during acute toxicity, increase in F2 isoprostane levels after toxicity, and increases in oxidized phospholipids occurring slightly later. Progressive increases in F2 isoprostanes and caspase 3/7 prior to and/or during acute toxicity suggests MTX induces oxidative stress and an associated increase in apoptosis. Since PC and PI comprise the cellular membrane, perhaps this slight delay in increased oxidized PC and PI levels reflect effects of oxidative stress and induction of apoptosis. For all biomarkers, increases were greatest in Case 1; Case 1 had the most serious and persistent acute MTX toxicity. Normal ranges in healthy children for these biomarkers are not available. However, postinduction biomarker values in ALL patients undergoing treatment without evidence of neurotoxicity from the larger longitudinal studies are found in Table 2. Values for postinduction phase were chosen because clinical presentation of MTX toxicity in all 3 cases occurred during this time. For Cases 1 and 2, biomarker levels are higher compared to ALL patients without neurotoxicity. Although biomarkers for Case 3 do not differ from this patient population, the values are much higher relative to her pretreatment levels. Overall, Case 3 had lower values for all biomarkers evaluated and less toxicity from pretreatment compared to the other 2 cases. A well-known consequence of oxidative stress is oxidation of lipids, referred to as lipid peroxidation (Milne et al., 2005; Roberts & Morrow, 2000). Lipid peroxidation is a self-sustaining process capable of extensive tissue damage (Pilitsis et al., 2002; Rouse, Nwokedi, Woodliffe, Epstein, & Klimberg, 1995). White matter has a high
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Taylor et al. lipid content (lipid:protein, 70:30) compared to other membranes, making it a preferential target for oxidative stress and lipid peroxidation (Verstraeten, Golul, Keen, & Orteiza, 1997). White matter changes are linked to longterm visual-motor skills, attention, general intelligence, and academic abilities in children previously treated for ALL or brain tumors and those with TBI (Mulhern et al., 2004; Reddick et al., 2003, 2006; Serra-Grabulosa et al., 2005). Smaller white matter volumes were significantly associated with larger deficits in attention, intelligence, and academic achievement in children who were on average 6 years from ALL diagnosis (Reddick et al., 2006). These 3 case studies confirm that the brain is particularly vulnerable to oxidative stress. These findings support the role of oxidative stress in MTX-related neurotoxicity. There are several limitations to this report. At pretreatment, some biomarkers were already elevated. This could be due to the leukemia itself causing an increase in oxidative stress and apoptosis. Another limitation is the changes observed on the MRIs have been reported in asymptomatic patients being treated for leukemia and may not be related to neurotoxicity. Lastly, normal ranges for these biomarkers in healthy children are not available for comparison.
Nursing Implications Pediatric oncology nurses play an important role in patient and family education and early detection of acute toxicity symptoms. Given that the overall cure rate for childhood leukemia is high, the specialty is uniquely poised to now increase our understanding of symptom toxicities during treatment for childhood leukemia. Why individual symptom differences occur will continue to be an important question to explore. As development of methods continue for oxidative stress and apoptosis biomarkers, they can be used to more fully understand mechanisms underlying treatment-related symptoms and individual susceptibility to oxidative stress, which both contribute to treatment success. Early identification of individual susceptibility to chemotherapy-induced oxidative stress and apoptosis will pave the way for investigation of neuroprotective strategies that will not compromise the goal of cure. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funded in part by the National Institute of Nursing Research Institute (R01NR010889)
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Rajamani, R., Muthuvel, A., Senthilvelan, M., & Sheeladevi, R. (2006). Oxidative stress induced by methotrexate alone and in the presence of methanol in discrete regions of the rodent brain, retina and optic nerve. Toxicology Letters, 165, 265-273. Reddick, W. E., White, H. A., Glass, J. O., Wheeler, G. C., Thompson, S. J., Gaijar, A., . . . Mulhern, R. K. (2003). Developmental model relating white matter volume to neurocognitive deficits in pediatric brain tumor survivors. Cancer, 97, 2512-2519. Reddick, W. E., Shan, Z. Y., Glass, J. O., Helton, S., Xiong, X., Wu, S., . . . Mulhern, R. K. (2006). Smaller white-matter volumes are associated with larger deficits in attention and learning among long-term survivors of acute lymphoblastic leukemia. Cancer, 106, 941-949. Richards, S., Pui, C. H., Gayon, P., & Childhood Acute Lymphoblastic Leukemia Collaborative Group (CALLCG). (2013). Systematic review and meta-analysis of randomized trials of central nervous system directed therapy for childhood acute lymphoblastic leukemia. Pediatric Blood and Cancer, 60,185-195. Roberts, L., & Morrow, J. (2000). Measurement of F(2)isoprostanes as an index of oxidative stress in vivo. Free Radical Biology and Medicine, 28, 505-513. Rouse, K., Nwokedi, E., Woodliffe, J. E., Epstein, J., & Klimberg, V. S. (1995). Glutamine enhances selectivity of chemotherapy through changes in glutathione metabolism. Annals of Surgery, 221, 420-426. Serra-Grabulosa, J. H., Junque, C., Verger, K., Salgado-Pineda, P., Maneru, C., & Mercader, J. M. (2005). Cerebral correlates of declarative memory dysfunctions in early traumatic brain injury. Journal of Neurology, Neurosurgery, and Psychiatry, 76, 129-131. Smith, K. J., Kapoor, R., & Felts, P.A. (1999). Demyelination: the role of reactive oxygen and nitrogen species. Brain Pathology, 9, 69-92. Stenzel, S. L., Krull, K. R., Hockenberry, M., Jain, N., Kaemmingk, K., Miketova, P., & Moore, I. M. (2010). Oxidative stress and neurobehavioral problems in pediatric acute lymphoblastic leukemia patients undergoing chemotherapy. Journal of Pediatric Hematology/Oncology, 32, 113-118. Uzan, M., Erman, H., Tanriverdi, T., Sanus, G. Z., Kafadar, A., & Uzun, H. (2006). Evaluation of apoptosis in
cerebrospinal fluid of patients with severe head injury. Acta Neurochirurgica, 148, 1157-1164. Varma, S., Janesko, K. L., Wisniewski, S. R., Bayir, H., Adelson, P. D., Thomas, N. J., & Kochanek, P. M. (2003). F2-Isoprostane and neuro-specific enolase in cerebrospinal fluid after severe traumatic brain injury in infants and children. Journal of Neurotrauma, 20, 781-786. Verstraeten, S. V., Golul, M. S., Keen, C. L., & Orteiza, P. I. (1997). Myelin is a preferential target of aluminum-mediated oxidative damage. Archieves of Biochemistry and Biophysics, 344, 289-294.
Author Biographies Olga A. Taylor, MPH, is a Senior Research Coordinator at Texas Children’s Cancer and Hematology Centers, Baylor College of Medicine. Marilyn Hockenberry, PhD, RN, PPCNP-BC, FAAN is the Bessie Baker Professor of Nursing and Professor of Pediatrics at Duke University. Kathy McCarthy, BSN, RN, is a Senior Research Nurse at Texas Children’s Cancer and Hematology Centers, Baylor College of Medicine. Patricia M. Gundy, MS, is a Principal Research Specialist and Biological Sciences Laboratory Manager in the College of Nursing at the University of Arizona in Tucson. Adam Ross, BS, is a graduate student at the University of Arizona. He was a laboratory technician responsible for the F2 isoprostane results included in this manuscript. David Montgomery, PhD, is a Research Professor, College of Nursing, University of Arizona, and a research scientist (without compensation appointment), Research Service, Department of Veterans Affairs, Veterans Affairs Medical Center, Tuscan, Arizona. Michael E. Scheurer, PhD, is Associate Professor in the Department of Pediatrics at Baylor College of Medicine and Director of the Epidemiology Center at Texas Children’s Cancer Center. Ida M. Moore, PhD, RN, FAAN, is the Anne Furrow Professor and Biobehavioral Health Science Division Director at the University of Arizona College of Nursing.
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research-article2015
JPOXXX10.1177/1043454214563409Journal of Pediatric Oncology NursingTaylor et al.
Case Studies
Evaluation of Biomarkers of Oxidative Stress and Apoptosis in Patients With Severe Methotrexate Neurotoxicity: A Case Series
Journal of Pediatric Oncology Nursing 1–6 © 2015 by Association of Pediatric Hematology/Oncology Nurses Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1043454214563409 jpo.sagepub.com
Olga A. Taylor, MPH1, Marilyn J. Hockenberry, PhD, RN, PNP, FAAN2, Kathy McCarthy, BSN, RN1, Patricia Gundy, MS3, David Montgomery, PhD4, Adam Ross, BS3, Michael E. Scheurer, PhD, MPH1, and Ida M. Moore, PhD, RN, FAAN3
Abstract Central nervous system (CNS) treatment is an essential part of acute lymphocytic leukemia (ALL) therapy, and the most common CNS treatment is intrathecal (IT) and high-dose intravenous (IV) methotrexate (MTX). Treatment with MTX may cause neurotoxicity, which is often accompanied by neurologic changes, delays in treatment, and prolonged hospital stays. This article reports clinical presentations of 3 patients with severe MTX toxicity as well as levels of oxidative stress and apoptosis biomarkers in cerebrospinal fluid (CSF). Oxidative stress was measured by oxidized phosphatidylcholine (PC), oxidized phosphatidylinositol (PI), and F2 isoprostanes; apoptosis was measured by caspase 3/7 activity. Most consistent biomarker changes in all 3 cases were increases in caspase 3/7 and F2 isoprostanes prior to acute toxicity while increases in oxidized phospholipids occurred slightly later. Progressive increases in F2 isoprostanes and caspase 3/7 activity prior to and/or during acute toxicity suggests MTX induces oxidative stress and an associated increase in apoptosis. These findings support the role of oxidative stress in MTX-related neurotoxicity. Keywords neurotoxicity, methotrexate, leukemia, oxidative stress
Introduction In the United States, acute lymphocytic leukemia (ALL) is the most prevalent cancer among children and adolescents less than 15 years of age (Howlader et al., 2013). Central nervous system (CNS)-directed treatment is an essential part of ALL therapy, and the most common CNS treatment is intrathecal (IT) and high-dose intravenous (IV) methotrexate (MTX), which are administered throughout ALL therapy (Richards, Pui, Gayon, & Childhood Acute Lymphoblastic Leukemia Collaborative Group [CALLCG], 2013). MTX increases oxidative stress in several organs, including the brain, and white matter is particularly vulnerable to oxidative stress because of its high lipid content (Inder et al., 2002; Rajamani, Muthuvel, Senthilvelan, & Sheeladevi, 2006). Recent studies confirm an association between oxidative stress levels in the cerebrospinal fluid (CSF), MTX, and CNS injury (Caron et al., 2009; Hockenberry et al., 2013; Protas, Muszynska-Roslan, Holownia,
Krawczuk-Rybak, & Braszko, 2010; Stenzel et al., 2010). This article reports clinical presentations of 3 patients with severe MTX toxicity as well as CSF levels of oxidative stress and apoptosis biomarkers. Oxidative stress was measured by oxidized phosphatidylcholine (PC), oxidized phosphatidylinositol (PI), and F2 isoprostanes; apoptosis was measured by caspase 3/7 (Illingworth & Glover, 1971; Iwai et al., 2003; Porter & Janicke, 1999; Smith, Kapoor, & Felts, 1999).
1
Baylor College of Medicine Houston, TX, USA Duke School of Nursing, Durham, NC, USA 3 University of Arizona College of Nursing, Tucson, AZ, USA 4 Southern Arizona VA Health Care System, Tucson, AZ, USA 2
Corresponding Author: Olga A. Taylor, MPH, Baylor College of Medicine, 1102 Bates Street Suite 1580 Houston, TX 77030, USA. Email: [email protected]
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Setting and Sample These 3 cases were part of a larger longitudinal study evaluating oxidative stress biomarkers in pediatric ALL patients undergoing chemotherapy. Subjects were from 2 cancer centers in the southwestern United States. Each of the center’s institutional review boards approved the study. Parents or legal guardians provided informed consent for all subjects, and subjects age 7 or older provided assent. Children were eligible for the study if they were diagnosed with ALL and being treated on Children’s Oncology Group Protocols, were between 2 years 9 months and 15 years of age at the time of diagnosis, and spoke English. Children with a preexisting medical history of neurological disorders (i.e., seizures), psychiatric disorders, or a traumatic brain injury associated with an alteration of consciousness or a developmental disability such as Down Syndrome were excluded. The patients included in this case series presented with neurological changes during treatment. Children with ALL received chemotherapy in 3 phases referred to as induction, postinduction, and continuation therapy. Induction therapy (1 month) included weekly treatment with vincristine and daunomycin (for high-risk ALL), a corticosteroid and a dose of PEG-asparaginase, and 2 IT MTX treatments. Postinduction therapy (6-8 mo) involved several courses of treatment that included asparaginase, high- or intermediate-dose IV MTX (depending on ALL protocol assignment), vincristine, doxorubicin, corticosteroid, cyclophosphamide, cytarabine, and mercaptopurine. Continuation therapy (2-3 years) consisted of daily mercaptopurine and weekly oral MTX, with monthly pulses of vincristine and a corticosteroid. Throughout therapy, study participants received CNS prophylaxis with standardized doses of IT MTX based on age: 10 mg for children aged 2 to 2.99 years, 12 mg for children aged 3 to 8.99 years, and 15 mg for children aged ≥9 years. The 3 cases presented in this article had no identified leukemia blasts in their spinal fluid at ALL diagnosis and were receiving treatment in the postinduction phase of therapy when the clinical presentation of MTX toxicity occurred.
Methods for CSF Measures of Oxidative Stress CSF samples were collected when children with ALL had scheduled lumbar punctures at the time of diagnosis and subsequently for administration of IT MTX. CSF samples were placed on ice immediately after collection and centrifuged for 10 minutes at 3000 rpm to remove any cellular debris. Processed CSF was stored at –80º C. Promega CaspaseGlo luminescence assay kits were used to measure caspase 3/7 activity as instructed by the manufacturer. Each sample was measured in duplicate wells of 96 well, half-area white plates, with a sample volume of 25
µl/well. Plates were read using a Perkin-Elmer Envision Xcite multilabel plate reader with a high-sensitivity luminometer. Caspase enzyme activity was determined by comparison to a standard curve of the purified caspase enzyme (ENZO) using linear regression analysis and data expressed as Units/Lcaspase activity. Glycerophospholipids were extracted from 1 ml of CSF, separated by normal phase high performance chromatography (HPLC). Concentration was measured using peak area (defined as the area proportional to the amount of compound that is present), which is a measure of concentration. F2 isoprostanes were measured by a competitive enzyme-linked immunoassay (ELISA) kit according to instructions (Cayman Chemical, https://www.caymanchem.com). For a full description of methods for F2 isoprostanes, please see previous publication (Hockenberry et al., 2013).
Case 1 A 13-year-old female was diagnosed with high-risk pre-B ALL. Two months after diagnosis, the patient received the first course of postinduction therapy, a phase of intensive CNS treatment, consisting of intravenous cytarabine (Ara-C), oral mercaptopurine (6-MP), and IT MTX. After completing this course of therapy, the patient exhibited neurological changes, slurred speech and unsteady gait (walking to one side), at home. A day later, she could not speak and had trouble chewing. Prior to this event, the patient received a total of 75 mg of IT MTX (5 standard doses per protocol at 15 mg/dose for patients 9 years of age or older). Upon examination of the patient in the emergency room, right-sided weakness was found, which was substantially worse than the left side. Two days after the first neurological event, progression in the patient’s altered mental status and ataxia was observed. These findings are consistent with further evolution of IT MTX toxicity, and systemic chemotherapy treatment was resumed. The patient continued on high-dose IV MTX and oral MTX per protocol but received no IT MTX. Magnetic resonance imaging (MRI) was performed and revealed multiple lesions in the white matter thought to be consistent with MTX toxicity. Two biomarkers of oxidative stress, oxidized PC and F2 isoprostanes, in the CSF increased from pretreatment to period of acute toxicity (Table 1). The highest levels of oxidized PI were observed after the acute toxicity (peak area 1 023 715; Table 1). The highest levels for all oxidative stress biomarkers were found after acute toxicity (Table 1). From pretreatment, oxidized PC had a 9-fold increase, oxidized PI had a 3-fold increase, and F2 isoprostanes had a 2-fold increase (Table 1). Caspase 3/7 activity (apoptosis biomarker) increased from pretreatment (12.56 Units/L) to the neurotoxicity episode (113.5 Units/L) and then dropped after toxicity (25.61 Units/L).
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Taylor et al. Table 1. Biomarkers of Oxidative Stress and Apoptosis. Case 1 2 3
Sample Phase
Days From Diagnosis
Pretreatment Prior to toxicity Acute toxicity Event After toxicity Pretreatment Acute toxicity Event Event After toxicity After toxicity Pretreatment Prior to toxicity Acute toxicity Event After toxicity After toxicity
0 35 42 46 106 16 30 40 42 77 85 0 44 51 58 65 114
Caspase 3/7 (Units/L) 12.56 67.95 113.5 — 25.61 66.62 60.89 — — 50.85 50.6 7.6 20.19 35.89 — 59.75 41.48
F2 Isop (pg/mL) 11.092 18.616 17.556 — 24.91 9.511 10.621 — — 13.467 12.92 4.245 4.956 6.527 — 7.142 5.86
Oxidized PC (peak area)
Oxidized PI (peak area)
17 188 15 217 27 727
321 851 934 069 158 119 — 1 023 715 674 921 180 871 — — 910 859 1 196 316 252 273 108 948 420 336 — 80 689 532 779
— 152 900 38 749 26 385 — — 20 252 20 596 8581 12 781 10 171 — 14 021 6377
Abbreviations: Isop, isoprostanes; PC, oxidized phosphatidylcholine; PI, oxidized phosphatidylinositol.
Case 2
Case 3
A 14-year-old male was diagnosed with high-risk ALL. One month later, 10 days after IT MTX administration, the patient experienced acute onset of mental changes. Prior to this event, the patient had received 2 IT doses of 15 mg of MTX per protocol. The patient presented with generalized weakness associated with diaphoresis, left greater than right upper extremity weakness, with temporary resolution. While hospitalized, the patient had a subsequent episode of generalized weakness, this time with right upper extremity greater than left upper extremity weakness. Two days after the initial onset of mental status changes, the patient experienced right-sided facial droop and mechanical aphasia as well as right upper and lower extremity hemiparesis. MRI revealed lesions in the white matter, which were consistent with MTX toxicity, and chemotherapy treatment was resumed without IT MTX. The patient received Capizzi IV MTX (begins with low dose then gradually increases as tolerated), oral MTX and was restarted on IT MTX 6 months following the neurotoxicity episode. Oxidized PI and F2 isoprostanes had an overall increase from pretreatment to after toxicity (Table 1). F2 isoprostanes had a slight increase in concentration from pretreatment to period of acute MTX toxicity (9.5 pg/mL to 10.62 pg/mL). The greatest increase in oxidized PI was observed after toxicity (peak area 1 196 316; Table 1). Caspase 3/7 activity did not change significantly during the time points evaluated.
A 12-year-old female was diagnosed with intermediate risk T-cell ALL. Approximately two months later, the patient experienced acute mental confusion, right-sided weakness, and facial drooping. Prior to the onset of neurologic symptoms the patient received two 15 mg MTX doses given IT on treatment days 7 and 14. In addition to IT, the patient received 1 dose of cyclophosphamide 13 days prior to acute neurologic symptoms, 8 doses of intravenous Ara-C (last dose was 4 days prior to neurologic symptoms), and 13 daily doses of oral 6-MP. CT scan and MRI did not reveal any brain lesions. There was approximately a 2-fold increase in oxidized PI from pretreatment to after the acute toxicity event (Table 1). The greatest increase in oxidized PI was observed in the CSF sample after the neurotoxicity event (peak area 532 779; Table 1). Caspase 3/7 activity and F2 isoprostanes concentration also increased progressively from pretreatment to acute toxicity (Table 1). F2 isoprostanes increased from 4.25 pg/mL during pretreatment to 7.14 pg/mL during acute toxicity. Caspase 3/7 activity had a more dramatic increase from 7.6 Units/L pretreatment to 59.75 Units/L during acute toxicity.
Discussion This case series is the first to our knowledge to examine CSF biomarkers of oxidative stress and apoptosis in patients with severe MTX neurotoxicity. These findings
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contribute to the growing body of evidence for the association of oxidative stress and effects of childhood cancer treatment. In this report, oxidized PC, oxidized PI, and F2 isoprostanes measured oxidative stress; apoptosis was measured by caspase 3/7 (Illingworth & Glover, 1971; Iwai et al., 2003; Porter & Janicke, 1999; Smith et al., 1999). F2 isoprostanes are formed from peroxidation of arachidonic acid, a polyunsaturated free fatty acid found in high concentrations in membrane lipids (Milne et al., 2005; Roberts & Morrow, 2000). In all 3 cases, F2 isoprostanes increased progressively prior to and/or during acute toxicity episodes. Varma et al. (2003) evaluated CSF F2 isoprostane levels in healthy children and those with traumatic brain injury (TBI). Pretreatment levels for the 3 cases reported here (range, 4.2-11.1 pg/ml; Table 1) are comparable to healthy controls (5.64 ± 8.08 pg/ml) reported by Varma et al. (2003). Values after acute toxicity, particularly for Cases 1 and 2 (24.91 pg/ml and 13.4 pg/ml, respectively; Table 1) are in the range of values found in children with TBI (15-36.59 pg/ml). These data suggest that MTX toxicity induces similar damage and increases in oxidative stress levels as children with TBI. Cell membrane phospholipids, such as PC and PI, have a high concentration of polyunsaturated fatty acids and are susceptible to oxidant attack (Girotti, 1998). In a sample of children with ALL (N = 36) who did not experience acute MTX toxicity, mean oxidized PC peak area pretreatment was 14 482.06 (SEM = 1193.41) (Hockenberry et al., 2013). The highest level of oxidized PC peak area occurred on day 8 of treatment (mean = 18 497.39; SEM = 2188.82). Oxidized PC peak area was approximately 8-fold higher in Case 1, and this patient experienced the most severe toxicity. This case study supports oxidized PC as a potential biomarker of oxidative stress in acute neurological toxicity. In this same sample of 36 children, mean oxidized PI peak area was 56 7977 (SEM = 11 8328) at diagnosis (Hockenberry et al., 2013). The greatest increase in oxidized PI also occurred on day 8 (mean = 902 886; SEM = 223 164). Although oxidized PI increased in all 3 cases, the increases were only greater in Cases 1 and 2 when compared to peak areas found in children without acute MTX toxicity. Caspase 3/7 activity is an established biomarker of apoptosis. Uzan et al. (2006) examined caspase 3 in the CSF of healthy patients and those with severe head injury. No detectable concentrations of caspase 3 activity were noted in controls. Caspase 3 activity in particular has been reported to be increased in traumatic brain injury (Harter, Keel, Hentze, Leist, & Ertel, 2001; Uzan et al., 2006). In Cases 1 and 3, CSF caspase 3/7 activity was elevated prior to (67.95 Units/L and 20.19 Units/L, respectively) and during acute neurotoxicity (113.5 Units/L to 35.89 Units/L, respectively). Of note, the
Table 2. Biomarkers in Acute Lymphocytic Leukemia (ALL) Patients Undergoing Treatment Without Evidence of Neurotoxicity. Therapy Phase Post induction
Caspase 3/7 Units/L (SEM)
F2 Isop pg/mL (SEM)
48.183 (1.99)
6.26 (0.34)
Oxidized Oxidized PI PC Peak Peak Area Area (SEM) (SEM) 14430 (500)
685 160 (96880)
Abbreviations: Isop, isoprostanes; PC, oxidized phosphatidylcholine; PI, oxidized phosphatidylinositol.
greatest increase was observed in Case 1 during the neurotoxicity event (113.5 Units/L). Although no dramatic changes were observed in Case 2, the concentration of caspase 3/7 activity was higher (range, 50.60-66.62 Units/L) than published controls and similar to that observed during acute toxicity in the other 2 cases. These patterns suggest increases in caspase 3/7 may precede increased oxidation of membrane phospholipids, perhaps due to its role in apoptosis activation. The most consistent biomarker changes in all 3 cases were increases in caspase 3/7 during acute toxicity, increase in F2 isoprostane levels after toxicity, and increases in oxidized phospholipids occurring slightly later. Progressive increases in F2 isoprostanes and caspase 3/7 prior to and/or during acute toxicity suggests MTX induces oxidative stress and an associated increase in apoptosis. Since PC and PI comprise the cellular membrane, perhaps this slight delay in increased oxidized PC and PI levels reflect effects of oxidative stress and induction of apoptosis. For all biomarkers, increases were greatest in Case 1; Case 1 had the most serious and persistent acute MTX toxicity. Normal ranges in healthy children for these biomarkers are not available. However, postinduction biomarker values in ALL patients undergoing treatment without evidence of neurotoxicity from the larger longitudinal studies are found in Table 2. Values for postinduction phase were chosen because clinical presentation of MTX toxicity in all 3 cases occurred during this time. For Cases 1 and 2, biomarker levels are higher compared to ALL patients without neurotoxicity. Although biomarkers for Case 3 do not differ from this patient population, the values are much higher relative to her pretreatment levels. Overall, Case 3 had lower values for all biomarkers evaluated and less toxicity from pretreatment compared to the other 2 cases. A well-known consequence of oxidative stress is oxidation of lipids, referred to as lipid peroxidation (Milne et al., 2005; Roberts & Morrow, 2000). Lipid peroxidation is a self-sustaining process capable of extensive tissue damage (Pilitsis et al., 2002; Rouse, Nwokedi, Woodliffe, Epstein, & Klimberg, 1995). White matter has a high
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Taylor et al. lipid content (lipid:protein, 70:30) compared to other membranes, making it a preferential target for oxidative stress and lipid peroxidation (Verstraeten, Golul, Keen, & Orteiza, 1997). White matter changes are linked to longterm visual-motor skills, attention, general intelligence, and academic abilities in children previously treated for ALL or brain tumors and those with TBI (Mulhern et al., 2004; Reddick et al., 2003, 2006; Serra-Grabulosa et al., 2005). Smaller white matter volumes were significantly associated with larger deficits in attention, intelligence, and academic achievement in children who were on average 6 years from ALL diagnosis (Reddick et al., 2006). These 3 case studies confirm that the brain is particularly vulnerable to oxidative stress. These findings support the role of oxidative stress in MTX-related neurotoxicity. There are several limitations to this report. At pretreatment, some biomarkers were already elevated. This could be due to the leukemia itself causing an increase in oxidative stress and apoptosis. Another limitation is the changes observed on the MRIs have been reported in asymptomatic patients being treated for leukemia and may not be related to neurotoxicity. Lastly, normal ranges for these biomarkers in healthy children are not available for comparison.
Nursing Implications Pediatric oncology nurses play an important role in patient and family education and early detection of acute toxicity symptoms. Given that the overall cure rate for childhood leukemia is high, the specialty is uniquely poised to now increase our understanding of symptom toxicities during treatment for childhood leukemia. Why individual symptom differences occur will continue to be an important question to explore. As development of methods continue for oxidative stress and apoptosis biomarkers, they can be used to more fully understand mechanisms underlying treatment-related symptoms and individual susceptibility to oxidative stress, which both contribute to treatment success. Early identification of individual susceptibility to chemotherapy-induced oxidative stress and apoptosis will pave the way for investigation of neuroprotective strategies that will not compromise the goal of cure. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funded in part by the National Institute of Nursing Research Institute (R01NR010889)
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Author Biographies Olga A. Taylor, MPH, is a Senior Research Coordinator at Texas Children’s Cancer and Hematology Centers, Baylor College of Medicine. Marilyn Hockenberry, PhD, RN, PPCNP-BC, FAAN is the Bessie Baker Professor of Nursing and Professor of Pediatrics at Duke University. Kathy McCarthy, BSN, RN, is a Senior Research Nurse at Texas Children’s Cancer and Hematology Centers, Baylor College of Medicine. Patricia M. Gundy, MS, is a Principal Research Specialist and Biological Sciences Laboratory Manager in the College of Nursing at the University of Arizona in Tucson. Adam Ross, BS, is a graduate student at the University of Arizona. He was a laboratory technician responsible for the F2 isoprostane results included in this manuscript. David Montgomery, PhD, is a Research Professor, College of Nursing, University of Arizona, and a research scientist (without compensation appointment), Research Service, Department of Veterans Affairs, Veterans Affairs Medical Center, Tuscan, Arizona. Michael E. Scheurer, PhD, is Associate Professor in the Department of Pediatrics at Baylor College of Medicine and Director of the Epidemiology Center at Texas Children’s Cancer Center. Ida M. Moore, PhD, RN, FAAN, is the Anne Furrow Professor and Biobehavioral Health Science Division Director at the University of Arizona College of Nursing.
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