Brain Imaging Changes with Melatonin Administration in Survivors of Childhood Hodgkin Lymphoma : A Report from the St. Jude Lifetime Cohort Aditya D. Mahadevan University of Florida Department of Applied Physiology and Kinesiology Nicholas Phillips, MD/PhD, Department of Epidemiology and Cancer Control, St. Jude Kevin R. Krull, PhD, Depart ment of Epidemiology and Cancer Control, St. Jude
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 2 ABSTRACT PURPOSE: Explore the effects of melatonin administration on white matter microstructure in the brains of adult survivors of Hodgkin Lymphoma. PATIENTS AND METHODS: 16 patients were assessed using diffusion weighted (dMRI) and T1 weighted MRI scans before and after 6 months of nightly melatonin administration. Before and after treatment, patients were also administered the self reported Pitts burgh Sleep Quality Index (PSQI). Changes in cortical gray matter thickness, fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity of white matter were analyzed from these scans. Patients were stratified by treatment arm (melatonin or placebo ) as well as by insomnia improvement as determined by PSQI scores. Preliminary tract based spatial statistics ( TBSS ) analysis using the FSL software library was conducted temporally on the entire sample regardless of stratification RESULTS: Significant increases in AD, RD, and MD were visualized throughout the brain across all strata. These changes were especially pronounced in the right hemisphere and in the memory associated hippocampal and cingulate regions. Large increases in cortical thickness were also seen in the cohort that reported improved sleep as well as both the placebo and melatonin groups. Participants that received melatonin showed larger increases i n gray matter volume in hippocampal areas than they did in other temporal regions. CONCLUSIONS : Longitudinal changes in AD, RD and MD indicate a significant effect of either sleep improvement or melatonin administration. Increases were observed in all three measures, which can be indicative of edema. Changes in cortical thickness seem to be associated with improved sleep rather than melatonin L arge increases were seen in the placebo group while no changes were seen in patients that reported no sleep improvement. Melatonin was as predicted, associated with larger changes in hippocampal regions than in other temporal regions. INTRODUCTION Hodgkin Lymphoma (HL) prognosi s, especially in pediatric cohorts is improving rapidly In the United States, the 5 year survival of pediatric patients with HL is 97.7% as of 2017 1 These survival rates are unprecedented and easily the highest of all pediatric cancers, necessitating a gradual shift of reso urces towards post treatment quality of life improvement. Patients with Hodgkin are often given an ABVD chemotherapy ag ent anthracyclines and bleomycin while receiving thoracic radiation treatment. C ardiovascular and pulmonary concerns have shown a dose dependent relationship with applie d mantle field radiation dosage 2 Additionally, a nthracycline administration in conjunction with mantle field radiation led to a 2.83 x increased risk for heart failure relative to patients receiving only thoracic
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 3 radiation 3 and patients treated with bleomycin and radiation had a 37% overall c hance of developing pulmonary dysfunction persistent for at least three years 4 Many cardiovascular diagnoses, including hypertension, diabetes, and atherosclerosis have been linked to increased ischemic stroke risk as they increase in severity 5 Pulmonary impairment, specifically deficit s in F orced E xpirator y V olume and F orced V ital C apacity is also linked to increased likelihood of ischemic stroke 6 T he induction of th ese stroke risk factors in patients with HL also increases incidence of transient ischemic attacks (TIA). Thes e are non infarctive and thus can often go unnoticed by the patient, but still provide the cerebral oxidative stresses and hypoxia, at lower levels, that are present after full scale ischemic stroke. This has been demonstrated in mice models Speci fically after induction of cerebral artery occlusion, mice exhibited large increases in many stress and heat shock proteins including hsp 72, hypoxia inducible factor 1 alpha, ubiquitin, and heat shock cognate protein 73 7 Treatment modalities can increase the risk of these incidents, as p atients with HL have also been shown to have a 4.4x higher incidence of stroke co mpared to sibling cohorts ; considerably higher in the presence of diabetes or hypertension. Given the lack of direct radiation to the CNS in these patients, it is evident that th is cerebrovascular dysfunction is a result of the vascular and pulmonary damag es induced by treatment 8 Leukoencephalopathy is a pattern of smal l white matter hyperintensities, generally indicative of cerebrovascular microtrauma and perfusive i ssues like the ones resulting from these cardiopulmonary morbidities in patients with HL In survivors of Hodgkin Lymphoma at least 15 years since diagnosis, presence of leukoencephalopathy showed a dose dependent re lationship with radiation, with 53% of s urvivors display ing leukoencephalopathy 2 Periventricular leukoencephalopathy has been shown to incre ase with decreased perfusion, decreased FEV(1)/FVC, and chronic obstructive pulmonary disease 9,10 The presence of many known cardiovascular morbidities and stroke risk factors such as hypertension, diabetes atrial fibrillation, a nd ventricular hypertrophy, have also been consistently
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 4 associate d with higher incidence of leukoencephalopathy, corroborating the theory of a vascular origin of thes e white matter changes 5,11,12 Increased CNS oxidative stress, as is induced by TIA and possibly by cardiovascular morbidity, has also been linked to oligodendrocyte toxicity, indicating reduced white matter microstructural quality 13 Hypertensive posterior cerebral leukoencephalopathy has also been shown to be reversible if patients are given antihype rtensive agents, demonstrating that if the underlying issues are corrected, white matter toxicity might be reversed 14 Leukoencephalopathy is a non specific index of induced white matter change the more precise nature of which m ight be elucidated from dMRI (Diffusion weighted MRI) /DTI (Diffusion Tensor Imaging) analys e s Cancer survivors, especially those treated for HL present significant fatigue, daytime sleepiness, and sleep dysfunction relative to their siblings 15 Decreased slow wave (stage 3 or 4) sleep is associated with insomnia 16,17 and as the percentage of total sleep time spent in slow wave sleep decreases, risk of developing hypertension has been shown to increase proportionally. Overall sleep duration under 7 hours in young er patients was significantly correlated with higher cha nces of developing hypertension, coronary heart disease and diabetes; all known risk factors for stroke 18 20 I nsufficient sleep could also contribute to TIA induced white matter deficit and oxidative stress. Fractional Anisotropy is an effective index of white matter diffusion directionality 21 Sleep deprivation has been associated with decreased in this measure via decreasing axial diffusivity (AD) another dMRI ind ex of white matter microstructure Insomnia symptoms have been shown to increase chances of cerebrovascular events by up to 27% a figure that could be higher in survivors of Hodgkin who experience cardiovascular morbidity as well 2 2 ,2 3 Sleep deprivation in mammalian neu ronal models has also been shown to increase membrane phospholipase Pla2g4c transcription, further suggesting weakness and breakdown of the plasma membrane 2 4 Sleep related g enes in general and specifically in oligodendrocytes have been strongly associated with myelination, cell proliferation, and phospholipid synthesis 2 5 2 6 Sleep deprivation of any amount has also been associated with increased concentrations of the protein hs CRP, which has been
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 5 identified as a stroke risk factor and agent of endothelial inflammation 2 7 Given this, it is possible that increased sleep could result in improved axial diffusivity and decreased R adial D iffusivity the latter of which has been linked to increased myelination 2 8 Melatonin is known to be the primary modulator of the sleep wake cycle and circadian rhythm as it increases in concentration as we fall into deeper sleep M elatonin administration has been shown to treat insomnia symptoms and thus increase sleep quality 2 9 In addition to insomnia attenuation, melatonin has been shown to act as a powerful scavenger of free reacti ve oxygen and nitrogen radicals recruiter of antioxidant enzymes including GPx and GRd and inhibitor of oxidative enzymes lik e quinone reductase 2 3 0 3 1 It has also been found in higher concentrations in the cerebrospinal fluid ( CSF ) allowing its effect to be more pharmacodynamically efficient in CNS inflammation including that resulting from TIA and infarctive incidents 3 2 Melatonin is released at peak concentration during deeper slow wave and RE M sleep. One of the most common reports from patients following melatoni n is vivid and frequent dreamin g This is indicative of an increase in REM sleep activity 3 3 which along with melatonin itself 3 4 has been heavily connected to increased activit y in the hippocampus 3 5 the known White Matter Change Treatment Sequela Pulmonary and Vascular Dysfunction Hypoxia and Perfusive Impairment Fatigue and Insomnia Stroke Risk Figure 1: Model of i nduced w hite m atter c hange in l ong term s urvivors of Hodgkin Lymphoma
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 6 initiator of memory formation Memory problems in patients with HL have been associated with decreased cortical gray matter thickness in temporal regions 2 Melatonin has been experimentally shown to decrease hypoxic oxidative stress in the hippocampus by decreasing release of Nitric Oxide and increased Ca2+ activated K+ channels as well as by its known free radical scavenger action 3 6 ,3 7 a possible treatment for these memory problems. Given t he highly inflammatory state of the CNS in patients with HL and the outstanding anti inflammatory effects of melatonin both before and after cerebrovascular events it is possible that treatment with melatonin will lead to more si gnificant white matter changes than sleep enhancement alone Given this, the largest improvements in white matter microstructure should be visualize d in the hippocampus both directly by antioxidant effect and indirectly b y increasing REM sleep activity. This should be accompanied by larger increases in hippocampal cortical thickness than in overall temporal cortical thickness METHODS Sample 17 survivors of Hodgkin Lymp homa w ere enrolled concurrently in the MIND and BRIGHT studies All survivors provided written informed consent and the protocol was approved by the institutional review board Inclusion criteria required only that the patients were enrolled in St. Jude Life, had an FSIQ (Full Scale Intelligence Quotient) score of 79+, were 10+ years from diagnosis, and that they were at least 18 years of age. Patients were excluded if they we re already taking melatonin, displayed any true sleep disorder (apnea, RLS, etc.), had diabetes, worked nights, or reported seizures or illicit drug use. Impairment prior to treatment, pregnancy, or history of H igh D ose M ethotrexate (HDMTX) or C ranial R a diation T herapy (CRT) were also criteria for exclusion. One patient was later excluded after follow up led to a thyroid cancer diagnosis. The 16 remaining survivors were uniformly Caucasian and included
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 7 nine male and seven female participants. Mean age at diagnosis was 14.8 years, the highest and lowest ages being 19.9 and 6.6 respectively. Average age at assessment was 36 years. Procedure Survivors self reported sleep quality via the Pittsburg Sleep Quality Index (PSQI). Structural MR imaging included a T1 weighted image set acquired using sagittal 3D MPRAGE sequence (TR/TE/TI = 1980/2.32/1100ms) with an imaging r esolution of 1.0 mm isotropic Five participants were administered a nightly 3mg melatonin intervention and e leven were administered a similarly regimented placebo After 6 months, patients were again assessed via dMRI and PSQI administration. Insomnia was defined as a PSQI value of 3 for either measures of latency or maintenance and sleep improvement was defined as neither of these values exceeding 2 after intervention. Eleven patients presented insomnia symptoms and seven of th e se patients reported sleep improvement upon follow up. Diffusivity changes in DTI elucidate more specific changes in the white matter structure and integrity W hite matter health or toxicity can be visual ized on DTI as changes in Axial Diffusivity (AD) Radial Diffusivity (RD) Mean Diffusivity (MD) or Fractional Anisotropy (FA) MR diffusion tensor imaging (DTI) was acquired using bipolar diffusion encoding gradients to reduce gradient induced eddy currents using a double spin echo, echo planar imaging pulse sequence (TR, 14,800 ms; TE, 120 ms, b = 700 s/mm 2 ). Seventy or more 2 mm thick images were acquired in contiguous axial sections to provide whole brain coverage. Using a 128 square matrix, the 220 mm field of view provided an in plane resolution of 1.7 mm 2 Two acquisitions of thirty one image set s were collected. The thirty one image sets consisted of one in which b = 0 and 30 noncollinear, noncoplanar diffusion gradient directions in which b = 700 s/mm 2 to calculate the diffusion tensor for each voxel.
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 8 Statistical Analysis Voxelwise statisti cal analysis of the AD, RD, MD, and FA was conducted by using Tract Based Spatial Statistics (TBSS) analysis 3 8 in FMRIB Software Library ( FSL ) v5.0 3 9 The mean images for each index were skeletonized using a brain extracted mask 40 and co mpared to each subject aligned data via voxelwise cross subj ect statistics. Cortical gray matter thickness values were also abstracted from the structural images using the FMRIB Software Library (FSL) Scans were first converted from DICOM to NIFT I, removing images affected by large motion induced artifact. A brain mask extraction was conducted using BET and then DTI scans were processed using the dtifit tool. The principal eigenvector (V1) was then checked for anatomical alignment and the FA and M D maps were checked for blatant errors. After the AD and RD maps were similarly computed and checked TBSS analysis was run, followed by higher level statistical analysis using FEAT for multiple regression based data modeling and 5000 permutation randomiza tion with multiple comparison correction Preliminary analysis was conducted on the pre and post intervention scans from all participants to elucidate temporal changes in the entire sample. Resulti ng data for FA, AD, MD, and RD wer e compared across brain regions. Volume based, T1 derived c ortical thickness data was also analyzed bilaterally before and after treatment across all strata, which showed significant change in the following hippocampal and temporal subfields: The 4 Cornu Ammonis (CA) areas, the fimbria, hippocampal fissure, presubiculum, subiculum, entorhinal cortex, fusiform gyrus; inferior, middle, and superior temporal gyri; parahippocampal gyrus, transverse temporal gyrus, and temporal pole These areas were then analyzed according to treatme nt arm and sleep improvement grouping. Paired t tests were used to assess which subfields experienced temporal change in thickness throughout the sample followed by the treatment effect test and sleep improvement stratification test for those fields. Stat istical significance was defined as a t statistic of at least 1.5 as many of the associated p scores would move toward significance in a larger sample.
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 9 Statistical data was filtered to show only areas with a t statistic from between 1.5 and 6 to elimin ate outliers and no n significant data RESULTS Initial DTI analysis found significant temporal increases in AD, MD, and RD throughout the sample, indicating that a tem poral effect indeed exists regardless of treatment arm or sleep improvement However, it also revealed some artifact in the FA images as many voxels presented FA values greater than 1. Smoothing this data greatly reduced this present noise maki ng the diffusivity changes more apparent, but also lead ing to a significant decrease in analysis sensitivity. After preliminary pre stratified longitudinal analysis of all patients, the smoothed data presented only AD results that were strong Figure 3 Smoothed Figure 2 Unsmoothed Figure s 2&3 : Effect of smoothing on single patient (top) and composite (bottom) FA images. Images prior to smoothing are shown in Figure 2 and after smoothing in Figure 3 Figure 4: Axial Diffusivity results after smoothing and TBSS analysis Figure 4 Smoothed Axial Diffusivity
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 10 enough to remain significant (t > 1.5). Alternatively, to eliminate noise while preserving sensitivity, the FA skeleton threshold was increased from 0.2 to 0.3 to exclude noisy data from the gray matter and restrict the skeleton to the midline of white matter tr acts. After TBSS visualized increases in AD, RD, and MD were retained (t > 1.5). Multiple comparison correction to limit false positives also led to the loss of some large clusters of seemingly substantial white matter change in the brain stem. This loss of data is likely secondary to the extremely limited sample size used, which increased the likelihood of a false negative result post correction. Figures 5, 6, & 7: Increases in RD (Figure 5 ), MD (Figure 6), and AD (Figure 7) without smoothing after increasing skeleton threshold. Figure 6 Mean Diffusivity F igure 7 Axial Diffusivity Figure 5 Radial Diffusivity
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 11 Table 1 : Largest changes in Radial Diffusivity Figures 8 & 9: Effects of Multiple Comparisons Correction. Figure 8 shows composite image prior to correction and Figure 9 shows images following correction. Figure 8 FA Image before MC Correction Figure 9 MC Corrected FA Image
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 12 Abstracted data showed no areas of increased cortical gray matter thickness in patients that reported no improvement on their insomnia. Those who did report improved sleep showed increased cortical thickn ess in the Left CA4/Dentate Gyrus (t = 1.63), Right Inferior Temporal Lobe (t = 2.05), Right Middle Temporal Lobe (t = 2.77), Right Parahippocampal Gyrus (t = 3.4 4), and Right Fusiform Gyrus (t = 2.85). Subjects receiving the placebo sho wed improvement in 4 areas: the Left Subiculum (t = 2.46), Right Middle Temporal Gyrus (t = 1. 70), Left CA4/Dentate Gyrus (t = 1.79), Right Parahippocampal Gyrus (t = 2.1) In the melatonin group, gray matter thickness was increased more significantly i n the Right Hippocampal Entorhinal Cortex (t = 3.4) and Right Parahippocampal Gyrus (t = 2.1) than in the superior (t = 1.6), middle (t = 1.7), and inferior (t = 1.6) gyri. Table 2 : Largest changes in Axial Diffusivity Figure 10 : Visualized changes in cortical thickness in all subjects that reported improved sleep following either melatonin or placebo treatment. Sleep Improvement
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 13 DISCUSSION Increases in AD, RD, and MD were especially strong in the right hemisphere which is more strongly implicated in memory storage and formation than the left Increases in both AD and RD were seen in the cingulate gyrus which, like the hippocampus, has been implicated in memory formation among other functions G lobal i ncreases in AD generally indicate heightened white matter quality while increases in RD are interpreted as the opposite In order to truly elucidate the white matter effect of Figure 11 : Visualized changes in cortical thickness in all subjects that received the melatonin treatment. Melatonin Placebo Figure 12 : Visualized changes in cortical thickness in all subjects that received the placebo treatment.
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 14 melatonin administration, a larger and more powerful study must be conducted as our sample was limited, and a fairly liberal t value threshold was applied C ortical thickness values showed significant increases in the sleep improvement cohort, while no such increases were seen in those who report ed no improve ment in sleep. S imilar increases were observed in both the melatonin and placebo groups This indicates that the changes seen could be due in larger part to improved sleep and not the anti oxidant capacity of melatonin It could also in initiated cascade that can be reparative for white matter. In the melatonin group, larger gray matter thickness increases were seen in the hippocampal regions than in the temporal regions. This align s with the idea that the increas ed REM sleep and dreaming seen with melatonin administration is in part related to heightened hippocampal activity. The data show changes in white matter integrity and gray matter thickness resulting from melatonin administr ation, and if further analyses can show this to be a positive clinical effect, melatonin could be explored as an adjunctive treatment during radiation and chemotherapy to ameliorate their late effects. Anthracycline induced cardiovascular damage may be due to the deeply investigated anthracycline induced hyperactivity of Nitric Oxide Syntha se and production of peroxynitri te radicals from NO that remains unbound to the endothelium. These radicals cause oxidative endothelial damage and lead to an increase in cytosolic calcium levels that can be toxic to mitochondria and lead to apoptosis 41 This causes a quasi atherosclerotic effect in the vasculature that could contribute significantly to any perfusion related white matter change and the resulting neurocognitive deficit seen in patients Radiation has been shown to lead to similar effects in the irradiated vasculature. In irradiated endothelium, increased leukocyte and platelet adhesion molecules, including CD31, have been consistently observed 4 2 ,4 3 leading to a similar inflammatory and fibrotic effect as the one caused by anth racycline administration. After just weeks of radiation, significant fibrosis and luminal narrowing have been observed in surgical studies 4 4 Bleomycin and radiation affect the lungs similarly by inhibiting the type II epithelial cell proliferation that normally induces re
White Matter Effects of Melatonin in Survivors of Childhood Hodgkin Lymphoma 15 epithelialization after injury to type I cells. This leads to a sustained inflam matory response 4 5 Bleomycin is associated with increases in TGF and IL 4, markers of increased macrophage and T cell activity respectively 4 6 ,4 7 These inflammatory ma rkers recruit fibroblasts, which secrete collagen in to the alveolar space causing fibrosis and decreased lung elasticity, ultimately impairing function. In rat models, one instance of 28 Gy thoracic radiation has been associated with systemic hypoxia due to damage to pulmonary vasculature. This hypoxia was followed by an increase in oxidative species concentration and macrophage activity, indicating a large inflammatory response. Bleomycin and radiation induced hypoxia co uld also exacerbate hypoxic inducti on of leukoencephalopathy 4 8 Melatonin has also been shown to decrease the effect of calcium calmodulin, thereby increasing the polymerization of microtubules 4 9 This causes a decrease in smooth muscle contraction 50 a natur al dilatory effect that decrease s NO synthase activity 51 and could mitigate anthracycline induced NO synthase hyperactivity. Melatonin has also been shown to attenuate the concentration of reactive species and the inflammation induced by anthracycline administration while slow ing atherosclerotic progression vascular aging, and endothelial inflammatory processes in mammals 52 5 4 Melatonin has al so been shown to protect against middle cerebral artery occlusion, decreasing chances of TIA or stroke. Melatonin has also been shown to decrease effects of ischemia reperfusion injury, attenuate the increase in blood brain barrier permeability seen after ischemic stroke, and augment neural electrical responsiveness after hemorrhagic stroke 5 5 5 7 indicating potential use as pr eventative or reparative drug for stroke. Given the white matter effect shown in the imaging data of these patients, melatonin could be explored as an adjuvant treatment to prevent lung dysfunction and proliferation of vascular ROS and RNS species. While melatonin is present in lower quantities in the systemic cardiova scular system than it is in the CNS, it is feasible that it can have a significant enough effect to decrease the atherosclerotic behavior and lung inflammation that causes morbidities in th ese systems in survivors of HL. Melatonin has also been shown to de crease angiogenesis via inhibition of vascular endothelial growth factor and
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