Hets 2021

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Genetics of multiple smaller sample size for the NonMS cohort. This issue sclerosis. Biochim Biophys Acta 2011;1812:194–201. should also be addressed in future studies. 9. International Multiple Sclerosis Genetics C, Wellcome In this study, we have focused on diagnostically chal- Trust Case Control C, Sawcer S, et al. Genetic risk and a lenging patients representing a true diagnostic dilemma, primary role for cell-mediated immune mechanisms in and present a novel MS diagnostic tool that can be used multiple sclerosis. Nature 2011;476:214–219. at the early disease stage to assist in accurate diagnostic decision making. 10. International Multiple Sclerosis Genetics Consortium. Network-based multiple sclerosis pathway analysis with Acknowledgments GWAS data from 15,000 cases and 30,000 controls. Am J Hum Genet 2013;92:854–865. This study has not received any private or public funding. 11. Sawcer S, Franklin RJ, Ban M. Multiple sclerosis genetics. Author Contributions Lancet Neurol 2014;13:700–709. Michael Gurevich, Ph.D. – contributed to study design, 12. Shastry BS. SNPs: impact on gene function and phenotype. analysis of data, and manuscript drafting and revising. Methods Mol Biol 2009;578:3–22. Gadi Miron, M.D. – contributed to study design, analysis of data, and manuscript drafting and revising. Anat 13. R development Core Team. R: a language and Achiron, M.D., Ph.D. – contributed to study design environment for statistical computing. Vienna, Austria: R analysis of data and manuscript drafting and revising. foundation for Statistical Computing, 2013. Conflict of Interest 14. Piccolo SR, Sun Y, Campbell JD, et al. A single-sample microarray normalization method to facilitate Dr. Achiron, Dr. Gurevich and Dr. Miron has a patent personalized-medicine workflows. Genomics 2012;100:337– US Prov 61/974,455 filed on 03/04/2014 pending. 344. 15. Johnson WE, Li C, Rabinovic A. 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Injury to white matter tracts in relapsing-remitting multiple sclerosis: A possible therapeutic window within the first 5 years from onset using diffusion-tensor imaging tract-based spatial statistics NeuroImage Clinical | 2015 ‫ פרופ' ענת אחירון‬:‫מנחה‬ ‫ מנהלת המרכז‬,‫מייסדת פרויקט ח\"ץ‬ ‫לטרשת נפוצה ואחראית הקתדרה‬ ‫למחלות אוטואימוניות אוניברסיטת ת\"א‬ [email protected] ‫אסף אחירון‬ ‫אונ' תל אביב‬ ‫השתתף כסטודנט בפרויקט ח״ץ‬ 2007-2009 ‫בין השנים‬ [email protected] 201

NeuroImage: Clinical 8 (2015) 261–266 Contents lists available at ScienceDirect NeuroImage: Clinical journal homepage: www.elsevier.com/locate/ynicl Injury to white matter tracts in relapsing–remitting multiple sclerosis: A possible therapeutic window within the first 5 years from onset using diffusion-tensor imaging tract-based spatial statistics Achiron Asaf *, Stone Evan, Achiron Anat Multiple Sclerosis Center, Sheba Medical Center, Tel-Hashomer, Ramat-Gan, Israel Sackler School of Medicine, Tel-Aviv University, Israel article info abstract Article history: DTI studies in multiple sclerosis (MS) reveal white matter (WM) injury that occurs with disease progression. In Received 22 February 2015 the present study we aimed to elucidate the relationship of microstructural WM damage in patients with varying Received in revised form 15 April 2015 periods of disease duration. DTI scans were acquired from 90 MS patients and 25 healthy controls. Patients were Accepted 28 April 2015 grouped to short (b1 year), moderate (1 up to 6 years) and long (6–10 years) disease duration periods. Statistical Available online 30 April 2015 analyses of the fractional anisotropy (FA) data were performed using tract-based spatial statistics (TBSS). Whole- brain skeletal FA measurements showed a significant decrease between healthy controls and the short MS dis- Keywords: ease duration group, as well as between moderate disease duration and long disease duration groups, but failed Multiple sclerosis to show a significant difference between short and moderate disease duration groups. White matter Voxelwise analysis revealed clusters of diffuse FA reductions in 40 WM tracts when comparing healthy controls Diffusion-tensor imaging (DTI) and MS short disease duration group, with the point of maximal significant difference located in the left inferior Fractional anisotropy (FA) longitudinal fasciculus. Tract-based spatial statistics (TBSS) Comparing short with long disease duration groups, progressive FA reduction was demonstrated across 30 WM tracts, with the point of maximal significant difference migrating to the body of the corpus callosum. A non-linear pattern of WM microstructure disruption occurs in RRMS. Alterations are seen early in the disease course within 1 year from onset, reach a plateau within the next 5 years, and only later additional WM changes are detected. An important period of a possible therapeutic window therefore exists within the early disease stage. © 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction tensor imaging (DTI) is a nonconventional MRI technique that is sensi- tive to the magnitude and direction of water diffusion within tissues and White matter (WM) injury is the cardinal pathological process oc- therefore can provide integrity assessment and measurements of WM curring early in multiple sclerosis (MS). Disruptions of WM tracts are tract damage even in the presumed normal appearing WM (Giorgio significant predictors for physical disability and cognitive impairment et al., 2010). in MS, and hence are important to be quantified at different stages of the disease course (Samann et al., 2012). WM damage can be detected DTI allows for calculation of the fractional anisotropy (FA), which is a and measured by brain magnetic resonance imaging (MRI), which dem- quantitative parameter for evaluating the direction-dependent diffusiv- onstrates focal and macroscopic inflammatory lesions by T2 and FLAIR ity of water molecules along the WM tracts. FA ranges from 0 to 1 and is as well as axonal loss by T1 black holes. However, injuries outside an indirect marker for WM integrity, as decreased FA reflects greater de- these lesions are not easily detected by conventional MRI techniques. myelination of fibers (Commowick et al., 2008; Roosendaal et al., 2009). Therefore, there is a need to apply innovative methods that may provide This is in accordance with a pathological study demonstrating that FA is additional insight into the ongoing pathological processes. Diffusion affected by myelin content in post-mortem MS brains (Schmierer et al., 2007). Aligning FA data from multiple subjects can be performed Abbreviations: MS, multiple sclerosis; WM, white matter; FA, fractional anisotropy; by tract-based spatial statistics (TBSS), an automated observer- TBSS, tract-based spatial statistics; EDSS, expanded disability status scale; RRMS, relaps- independent method. TBSS allows group-wise comparisons of DTI ing–remitting multiple sclerosis; CIS, clinically-isolated syndrome; ILF, inferior longitudinal data by analyzing it in a voxel-wise fashion and minimizes registration fasciculus. errors, and is an advantageous approach as it enables that maximal FA values for each subject will be directly compared at each point even if * Corresponding author at: Multiple Sclerosis Center, Sheba Medical Center, Tel- the fiber centers are not perfectly aligned (Smith et al., 2007). TBSS Hashomer, Israel. Tel.: +972 3 5303932; fax: +972 3 5305076. has already been validated as an effective way to evaluate the FA in E-mail address: [email protected] (A. Asaf). http://dx.doi.org/10.1016/j.nicl.2015.04.020 2213-1582/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 202

262 A. Asaf et al. / NeuroImage: Clinical 8 (2015) 261–266 MS, and several studies reported widespread WM damage in patients Review Committee of Sheba Medical Center, Tel-Hashomer, Israel and with clinically isolated syndrome (CIS), RRMS and primary progressive all subjects gave written informed consent. MS (Bodini et al., 2009; Raz et al., 2010a; Roosendaal et al., 2009). The significance of TBSS analysis of DTI data from MS patients was 2.3. MRI acquisition protocol established by studies that demonstrated that decreased FA correlated with increased disability by EDSS (Liu et al., 2012), lower cognitive per- All subjects underwent the same brain MR imaging protocol, per- formance and specifically for processing speed, visual and verbal work- formed on 3.0-T MR scanner (Signa, GE) using high-resolution 8 chan- ing memory (Yu et al., 2012b). However, TBSS studies assessing patients nel head coil. Data were obtained using the following sequences: at different stages of disease course are scarce. Therefore, the aim of the (1) 3D-FSPGR (1 × 1 × 1 mm voxel, TE = 2 ms, TR = 6 ms), (2) T2- present study was to investigate how changes in WM tracts relate to MS FSE (slice thickness 2.6 mm, TE = 102 ms, TR = 3500 ms), (3) FLAIR disease duration, as clarifying this relationship could give better insight (slice thickness 2.6 mm, TE = 122 ms, TR = 9502 ms, TI = 2375 ms), on the microstructure changes that occur at different stages of the dis- (4) 2D-FSPGR with contrast GD-DTPA (slice thickness 2.6 mm, TE = ease course. To this end, we investigated how structural WM disrup- 2 ms, TR = 250 ms). Axial DTI data were acquired along 31 independent tions based on fractional anisotropy (FA) data and tract-based spatial directions using a single shot echo-planar imaging sequence, (TE = statistics (TBSS) analysis were affected by MS disease duration within 76 ms, TR = 14,000 ms, b = 1000 s/mm2, FOV 256 × 256 mm, ma- a cross-sectional study design in MS patients with short, moderate trix 128 × 128). Two additional images without diffusion weighting and long disease duration periods. We first investigated the relationship (b = 0 s/mm2) were acquired. Axial images were acquired by contigu- of whole-brain skeletal FA measurements between healthy subjects and ous slices with 2.6 mm thickness. The slices were positioned to run par- MS patients with short disease duration of up to 1 year from onset as we allel to a line that joins the anterior commissure–posterior commissure hypothesized that disruption of WM tracts will be identified already at plane. During image acquisition the same image resolution and the the this early disease stage. Further analyses were performed grouping same localizer were used for T2 Flair and DTI series, to obtain similar patients by 5 year intervals to moderate (from 1 year and up to 6 years axial slice positions. duration) and long (from 6 to 10 years inclusive) disease duration pe- riods to assess the implications of WM disruption as the disease pro- 2.4. Image analysis and post-processing gresses. In addition, we performed voxelwise analysis to recognize clusters of FA reductions in various WM tracts and to identify associa- Specifications of the methods for DTI analysis were previously tions between regional WM changes that are detected in the early described in detail (Smith et al., 2007). In brief, DICOM files of the DTI phase of MS and if these are similar in pattern in patients with moderate acquisition were converted into a single multivolume NIFTI file (Neuro- and long disease duration periods. imaging Informatics Technology Initiative file) and transferred to a linux-based workstation. Analysis process with FMRIB Software Library 2. Materials and methods (FSL, http://www.fmrib.ox.ac.uk/fsl) was initiated with eddy-current correction (compensating for distortions and for simple head motion) 2.1. Study design and followed by automatic brain extraction. Diffusion tensors were then reconstructed by fitting a diffusion tensor model at each voxel of Retrospective, cross-sectional in a tertiary referral center. the diffusion images and FA values were generated. Whole-brain voxelwise differences between subjects were carried out using TBSS 2.2. Subjects (Smith et al., 2004; Smith et al., 2006). First, FA data of all subjects were transformed into 1 × 1 × 1 mm3 MNI152 common space by In total, MRI data obtained from 100 MS patients and 30 healthy sub- means of nonlinear registration. Then, the transformed FA images jects were analyzed. Data from some subjects were excluded from anal- were averaged to generate a mean FA image which was subsequently yses because of neurological comorbidity (N = 7), too many artifacts or skeletonized, representing tracts common to all of the subjects. In noise in the raw MRI data (N = 4), or absence of complete MRI data order to prevent inclusion of non-skeleton voxels, each subject3s aligned (N = 4). FA map was mapped onto the “mean FA skeleton” using a lower thresh- old of FA of 0.2 in order to exclude gray matter voxels. The approach of Consequently, 90 MS patients and 25 healthy subjects remained in carefully tuned non-linear registration, followed by a creation of a mean the study. All MS subjects met McDonald diagnostic criteria (Polman FA skeleton intends to face the cross-subject spatial variability effect et al., 2011) and were recruited from the MS database registry at the (Douaud et al., 2007). FA values were extracted for whole-brain analysis Sheba MS Center, Tel-Hashomer. Enrolled patients fulfilled the follow- and for major WM tracts according to the ICBM-DTI-81 WM labels atlas, ing inclusion criteria: (1) Age above 18 years; (2) RRMS disease course; which includes 48 labels created by hand segmentation of a standard- (3) MRI examination performed on 3.0 T including DTI protocol; (4) no space average of diffusion MRI tensor maps from 81 healthy subjects relapse or corticosteroid use within 4 weeks before study entry to avoid (Oishi et al., 2008). In addition, two fiber tracts for the inferior longitu- transient confounding effects on MRI; and (5) absence of another major dinal fasciculi from the JHU-White Matter Tractography atlas were in- medical disorder. Clinically stable patients were defined as patients cluded for greater spatial coverage (Mori et al., 2009). without relapses in the last year prior to be enrolled in the study Pa- tients were divided according to disease duration periods: short, up to 2.5. Statistical analysis 1 year from clinical onset, moderate, 1 year and up to 6 years from clin- ical onset, and long, 6–10 years from clinical onset. Disease duration was Region-of-interest (ROI) analysis was performed to initially evaluate calculated since the onset of the first symptomatology suggestive of MS. the magnitude of FA decreases within predetermined regions. ROI anal- All subjects underwent comprehensive neurological examination and ysis was calculated by splitting the 4D post-threshold skeletonized vol- disability was scored by the Expanded Disability Status Scale (EDSS) ume of each subject3s WM tract and repackaging them into group 4D by MS specialist neurologists. Data for age-matched normal healthy files. In order to achieve accurate inference of multiple comparisons subjects were obtained from the hospital radiology MRI Unit database, over space, voxelwise analysis was performed by the randomize which includes MRI data of healthy volunteers recruited from the local permutation algorithm from the FSL library, with 5000 permutations community and hospital staff. None of the healthy volunteers suffered and a cluster significance level of p b 0.05 using threshold-free from any neurological disease or took central nervous system active cluster enhancement (TFCE) (Keihaninejad et al., 2012). TFCE through medications. The study protocol was approved by the Medical Ethical the randomize script in TBSS was utilized to establish the volume of 203

A. Asaf et al. / NeuroImage: Clinical 8 (2015) 261–266 263 significant changes in the skeleton on both whole-brain and regional 3.2. TFCE voxelwise analyses level, using the aforementioned atlases. Relationship between FA and clinical parameters was evaluated by Spearman3s rank correlation Additional in-depth voxelwise analyses with TFCE to accurately con- test. trol for multiple comparisons over space are shown in Table 3. The re- sults strengthen the whole-brain mean skeleton FA findings. There 3. Results were diffuse changes between age-matched healthy subjects and RRMS patients with short disease, virtually no significant change be- Ninety RRMS patients, 66 females, mean age ± SE: 37.6 ± 1.0, and tween short and moderate disease duration groups (0 voxels in every 25 healthy controls, 14 females, mean age ± SE: 35.1 ± 2.2 years region), and diffuse areas of changes between long and moderate dis- were included in this study. As expected, patients in the short disease ease duration groups. Long and short disease duration groups were duration group were younger and with lower neurological disability compared in order to demonstrate the magnitude of the widespread as measured by the EDSS. The rate of patients treated by immunomod- FA reduction. This voxelwise analysis revealed 31% diffuse FA reduction ulatory drugs (IMDs) was 65.7% in the total sample population, and was spanning a skeletal area of 60,784 of 195,350 total voxels in 40 major similar between the disease duration groups. In the short group, 65.4% WM tracts when comparing healthy controls and the short disease du- were treated with IMDs, and similarly 65.2% and 66.6% were treated in ration group. The point of maximal significant difference was observed the moderate and long disease duration groups, respectively. In the in the left inferior longitudinal fasciculus. FA reduction of additional 22% short duration group, 70.6% were treated for more than 3 months, was shown in 30 major WM tracts when comparing the long and mod- whereas in the moderate and long disease duration groups, 100% and erate duration groups, affecting 42,212 of 192,208 skeletal voxels, with 91.7% of treated patients, respectively, received IMD treatment for the point of maximal significant difference migrating to the body of the more than 3 months prior to the MRI examination. These similar rates corpus callosum. of treatment between patients enabled us to compare the impact of DTI variables between groups. The pertinent demographic information 4. Discussion is shown in Table 1. DTI has proven to be an effective tool for providing a non-invasive 3.1. Whole-brain mean skeleton FA in-vivo investigation into WM tissue alterations unseen through con- ventional MRI (Yu et al., 2012a). Whole-brain mean skeleton FA measurements were significantly decreased in RRMS patients with short disease duration as compared In our current study, we used a region-of-interest approach to inves- with healthy controls (0.443 ± 0.023 vs. 0.465 ± 0.023, p b 0.001), tigate the temporal nature of injury to WM tracts in RRMS patients in re- as well as in patients with long disease duration as compared with pa- lation to disease duration. The strengths of our findings are related to tients in the moderate disease duration group (0.427 ± 0.020 vs. the robust methodology combining a high-resolution imaging protocol 0.443 ± 0.022, p b 0.01), but failed to show significant difference in with both voxelwise and ROI approaches. This approach allowed us to mean FA values between short and moderate disease duration groups characterize regional tract damage that occurred in large patient sample (p = 0.95), Fig. 1. Correlations of FA values in various ROIs with disease groups with varying disease duration. variables, e.g., disease duration and neurological disability by the EDSS for the whole group of MS patients are shown in Table 2. Decrease in We have confirmed the early occurring WM alterations also through FA values correlated with disease duration while no correlations were voxelwise analysis, which further established the decrease in FA values found with disability. Graphical presentation of the magnitude of between patients with short, moderate and long disease duration pe- these FA reductions with disease duration is demonstrated in Fig. 2 riods. The diffuse changes throughout the cerebral WM tracts were sig- and Supplementary Movies 1 and 2. nificant even in patients with very short disease duration of less than one year as compared with age-matched controls. These findings are in agreement with previous studies that showed decreased in FA in MS patients with short disease duration. Raz et al. compared 34 MS Table 1 All MS patients MS patients by disease duration groups Demographic and clinical variables of the studied populations. 90 Healthy subjects 66/24 37.6 ± 1.0 (20.1–68.7) Short (b1 y) Moderate (1–5.9 y) Long (6–10 y) 4.2 ± 0.2 N 25 (0.0–9.9) 26 46 18 Female/male 14/11 2.0 ± 0.1 18/8 35/11 13/5 Age 35.1 ± 2.2 (22.6–58.0) (0.0–6.5) 35.0 ± 1.7 37.2 ± 1.3 42.2 ± 2.6 33.4 ± 9.1 (20.1–51.7) (21.5–59.0) (28.2–68.7) Disease duration (18.8–58.8) 0.5 ± 0.1 4.5 ± 0.1 8. 7 ± 0.3 65.7 (0.0–0.9) (1.0–5.9) (6.0–9.9) EDSS 1.6 ± 0.3 1.9 ± 0.2 2.7 ± 0.4 (0.0–5.5) (0.0–6.5) (1.0–6.5) Age at onset 34.5 ± 1.7 32.8 ± 1.3 33.5 ± 2.5 (19.6–51.2) (18.8–55.6) (19.9–58.8) % IMD Tx 65.4 65.2 66.6 IMD, N (% of treated) 36 (61.0) 8 20 8 Interferons 12 (20.3) 9 4 3 Glatiramer acetate 3 (5.1) 5 1 IVIg 1 (1.7) 12/17 (70.6) 1 Natalizumab 53/59 (89.8) 0 30/30 (100) 11/12 (91.7) IMD Tx duration N 3 months 33 (71.7) 14 (77.8) N (%) Clinically stable, N (%) EDSS = Expanded Disability Status Scale; IMD = immunomodulatory drug; Tx = treatment; N = number. 204

264 A. Asaf et al. / NeuroImage: Clinical 8 (2015) 261–266 Table 2 Number of voxels in WM tracts with significant reduced FA values between groups. WM tracts Disease duration groups Short vs. Moderate Long vs. Long vs. healthy vs. short moderate short subjects Fig. 1. Mean whole-brain skeletal FA value comparison between study groups. L cerebral peduncle 331 0 169 380 R cerebral peduncle 342 0 95 354 patients within 3 months from the onset of first symptomatology to 16 L cingulum cingulate 238 0 414 healthy controls, identifying and demonstrating widespread changes in R cingulum cingulate 288 0 306 322 FA, especially in the corticospinal tracts and the corpus callosum (Raz L cingulum hippocampus 0 327 et al., 2010b). Similarly, we have demonstrated that early FA changes R cingulum hippocampus 13 0 2 occurred in the corticospinal tracts and in the corpus callosum; however L ant corona radiata 11 0 5 136 the most significant change with the onset of disease was observed in R ant corona radiata 1314 0 10 1122 the left inferior longitudinal fasciculus (ILF) that connects the occipital L post corona radiata 1199 0 822 895 lobe with the anterior part of the temporal lobe, running laterally and R post corona radiata 726 0 472 517 inferiorly to the optic radiation fibers. The ILF is associated with visual L sup corona radiata 779 0 452 681 content organization and the establishment of its emotional significance R sup corona radiata 1004 0 508 625 and plays an important role in social tasks requiring recognition of face Body, corpus callosum 975 0 747 747 emotion expression (Kleinhans et al., 2008). Sensory deficits are typical Genu, corpus callosum 2888 0 560 3104 of MS at onset and we have previously shown the occurrence of cortical Splenium, corpus callosum 1211 0 2813 1354 thinning of several sensory-related regions in the early disease stage L corticospinal tract 2134 0 1364 2152 (Achiron et al., 2013b). As the ILF is known to be associated with visual R corticospinal tract 0 1494 comprehension and visual memory (Chanraud et al., 2010), our findings L external capsule 0 0 88 suggest that the impairment in its structural integrity may affect the ca- R external capsule 41 0 0 44 pacity of MS patients to appropriately and in-time comprehend visual Fornix 1174 0 0 727 cues, even if no significant neurological disability is demonstrated. Dis- L inf cerebellar peduncle 1110 0 562 638 ruption of the ILF was already reported to occur in MS patients with av- R inf cerebellar peduncle 124 0 509 158 erage disease duration of 3.6 years (Roosendaal et al., 2009). Our results L ant internal capsule 0 153 122 show that not only are these changes the most significant among the R ant internal capsule 0 0 0 102 WM alterations, but that they also occur earlier than previously reported. L post internal capsule 0 0 0 266 R post internal capsule 193 0 0 283 Further comparison of RRMS patients with short disease duration L retrolenticular internal capsule 72 0 0 344 (up to 1 year) with the moderate disease duration (between 1 and R retrolenticular internal capsule 376 0 108 519 6 years) showed no significant differences between WM skeletons on L medial lemniscus 117 0 296 630 both whole-brain skeletal FA measurements and in-depth TFCE R medial lemniscus 686 0 657 567 voxelwise analyses. The finding that short and moderate disease dura- Mid cerebellar peduncle 617 0 528 100 tion groups did not show any FA differences is of interest. This suggests Pontine crossing tract 0 0 0 83 that WM disruption reaches a plateau during this 5 year interval. Simi- L post thalamic/optic radiation 0 0 0 853 larly, changes in FA were not detected over a period of a 1 year follow- R post thalamic/optic radiation 1 0 0 88 up in clinically-isolated MS patients (Raz et al., 2010b), and no signifi- L sagittal striatum 0 0 0 1042 cant FA changes were identified over time in RRMS patients by repeated R sagittal striatum 1060 0 990 1071 MRI scans within 5 years from onset (Giorgio et al., 2010). This may be L stria terminalis 1085 0 813 250 related to the concept of “brain reserve,” whereby the disease-induced R stria terminalis 472 0 280 362 insult is not significant enough to result in structural changes within L sup cerebellar peduncle 543 0 168 302 the WM tracts, reflecting the capacity to withstand deteriorating pro- R sup cerebellar peduncle 280 0 297 251 cesses and the ability to endure or recover quickly from the insult. The L sup front-occipital fasciculus 274 0 226 21 idea of this reserve against brain damage stems from repeated observa- R sup front-occipital fasciculus 0 0 0 15 tions showing no apparent relationship between the degree of brain pa- L sup longitudinal fasciculus 1 0 0 27 thology or damage and the clinical manifestations of that damage R sup longitudinal fasciculus 83 0 0 31 (Stern, 2002). In both the early and moderate disease duration groups, L uncinate fasciculus 0 0 0 973 patients bear greater capacity to withstand acquired white matter in- R uncinate fasciculus 958 0 648 1130 sults. Related to this observation, a recent study reported that both pa- L inf longitudinal fasciculus 1086 0 883 39 tients with radiologically-isolated syndrome (RIS) and relapsing– R inf longitudinal fasciculus 53 0 8 remitting MS had altered integrity of WM tracts as compared to normal 40 0 1 matched controls. However, in measuring WM tract integrity, RIS 1768 982 1467 patients lacked functional reorganization in key brain networks, 1167 948 1050 There are diffuse changes between controls and short disease group, virtually no change between short and moderate disease groups (0 voxels in every region), and diffuse areas of change between long and moderate disease groups. Long and short disease groups were compared in order to demonstrate the magnitude of the widespread FA reduction. suggesting a model of “functional reserve” which may become upregu- lated, with an adaptive or maladaptive role, only at a later stage. (Giorgio et al., 2015) In contrast, the comparison between the moderate (1–6 years) and the long disease duration (6–10 years) groups showed diffuse and sig- nificant changes involving almost all major WM tracts, with FA reduc- tion of 22% affecting 42,212 of 192,208 skeletal voxels, with the point of maximal significant difference migrating from the ILF to the body of the corpus callosum which is widely known to be involved in MS. Ac- cordingly, FA reductions appear to exhibit a non-linear time course with two “accelerations”. An acute, diffuse WM change within the first year following the initial clinical event, and another more gradual de- cline occurred as the disease approaches the 10-year mark. We hypoth- esize that in the early stage, the sharp decline in FA relates to diffuse alterations in the normal-appearing WM, whereas the more gradual 205

A. Asaf et al. / NeuroImage: Clinical 8 (2015) 261–266 265 Fig. 2. Comparison of FA differences in WM tracts between healthy subjects and MS patients according to disease duration. Significant clusters of reduced FA (p b 0.05) are shown in red (axial) and orange (3-dimensional) overlaid on a template image brain. A, C, Comparison between healthy subjects and MS patients with short disease duration (b1 year). B, D, Comparison between healthy subjects and MS patients with long disease duration (N6 years). decrease over a longer disease duration relates to subsequent Wallerian patients was significant only at disease duration greater than 5 years degeneration and axonal disruption. This progression signifies a point- (Achiron et al., 2013a). of-no-return, whereby the accumulating WM damage spreads expo- nentially and therefore makes the first 6 years an important time- Other studies also reported DTI changes in RRMS patients within this frame for immunomodulatory drug treatment. This possibility of a ther- time-frame. Roosendaal et al. (2009), studying regional DTI differences apeutic window stands for the period of time following MS onset in 30 RRMS patients with a mean disease duration of 3.6 years as com- whereby early treatment may favorably affect long-term disease out- pared with 31 age-matched controls, demonstrated FA decrease in var- come, and has been also suggested by the results of our previous ious brain regions including the fornices, the left corona radiata, the study where we demonstrated that cognitive impairment in MS inferior longitudinal fasciculus in both hemispheres, both optic radia- tions, and parts of the corpus callosum; Giorgio et al. (2010) used Table 3 TBSS cross-sectional analysis in 45 RRMS patients with 3 years of dis- Correlation between FA values in various ROIs with MS disease related variables. ease duration and demonstrated decreased FA in the splenium of the corpus callosum and along the pyramidal tract; Kern et al. (2011) eval- ROI Disease duration Age uated 25 MS patients with a mean disease duration of 4.8 years in com- parison to 16 controls and demonstrated a decrease in FA in the Fornix r = −0.31 p = 0.002 r = −0.24 p = 0.02 transcallosal hand motor fibers and corticospinal tracts. L cerebral peduncle r = −0.29 p = 0.004 r = −0.20 p = 0.05 R cerebral peduncle r = −0.30 p = 0.03 r = −0.24 p = 0.02 The therapeutic window opportunity is further intensified by the Genu, corpus callosum r = −0.23 p = 0.023 fact that the WM damage accumulates microscopically but is not appar- Splenium, corpus callosum r = −0.31 p = 0.003 ent clinically, as we did not find correlation with neurological disability. R corticospinal tract r = −0.44 p = 0.000 This is in accordance with previous study that also did not find correla- R medial lemniscus r = −0.24 p = 0.021 tions between mean FA values and EDSS (Raz et al., 2010a). L medial lemniscus r = −0.31 p = 0.003 R inf cerebellar peduncle r = −0.34 p = 0.001 Some limitations apply to this work. First, the cross-sectional R pos internal capsule r = −0.26 p = 0.013 nature of the study limited our ability to monitor the evolution of WM L pos internal capsule r = −0.23 p = 0.031 distraction on an individual basis. A longitudinal study will enable L sup corona radiata r = −0.27 p = 0.01 us to verify the results by following the same patients overtime. R sup corona radiata r = −0.24 p = 0.021 Second, we had relatively small sample size particularly for the long L pos corona radiata r = −0.28 p = 0.007 (6–10 years) disease duration group, and third we were not able to syn- L pos thalamic/optic radiation r = −0.26 p = 0.01 R pos thalamic/optic radiation r = −0.30 p = 0.003 chronize the MRI data to different IMDs3 treatment history and current R sup long fasciculus r = −0.25 p = 0.01 treatment regimens and thus we did not analyze potential medication R tapetum r = −0.34 p = 0.001 effects on WM pathology. This was related to the diversity in the type L tapetum r = −0.35 p = 0.001 of treatment (e.g., different IMDs), difference in treatment duration (e.g., different time-frames for each IMD) and treatment sequence EDSS did not correlate with any WM tract. order (e.g., difference in the choice of the first, second and third lines ROI = region of interest; FA = fractional anisotropy; EDSS = Expanded Disability Status of IMD). Scale; R = right; L = left; inf = inferior; pos = posterior; sup = superior. 206

266 A. Asaf et al. / NeuroImage: Clinical 8 (2015) 261–266 It is of importance to mention that we have focused on global aspects tractography study. Neuroimage 55 (3), 1169–1177. http://dx.doi.org/10.1016/j. of the microstructural WM damage in patients with varying periods of disease duration future studies will need to focus on more homogenous neuroimage.2010.10.07721056674. groups of patients to identify local properties of WM damage over time Kleinhans, N.M., Richards, T., Sterling, L., Stegbauer, K.C., Mahurin, R., Johnson, L.C., as well as to examine other diffusion metrics such as axial and radial dif- fusivities that may add additional useful information about the processes Greenson, J., Dawson, G., Aylward, E., 2008. Abnormal functional connectivity occurring in MS. in autism spectrum disorders during face processing. Brain 131 (4), 1000–1012. In conclusion, utilizing DTI with TBSS analyses, we have demonstrat- http://dx.doi.org/10.1093/brain/awm33418234695. ed the occurrence of widespread injury to WM tracts in RRMS patients. Liu, Y., Duan, Y., He, Y., Yu, C., Wang, J., Huang, J., Ye, J., Parizel, P.M., Li, K., Shu, N., 2012. We have subsequently shown that this structural damage appears in the very early stage of the disease with less than a year from onset. These al- Whole brain white matter changes revealed by multiple diffusion metrics in multiple terations reach a plateau within the next 5 years, and only later as the pathological process accumulates, additional WM changes are detected. sclerosis: a TBSS study. Eur. J. Radiol. 81 (10), 2826–2832. http://dx.doi.org/10.1016/j. An important therapeutic window therefore exists within the early dis- ejrad.2011.11.02222172535. ease stage. Mori, S., Oishi, K., Faria, A.V., 2009. 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‫‪Normal fetal posterior fossa in MR imaging:‬‬ ‫‪new biometric data and possible clinical significance‬‬ ‫‪American Journal of Neuroradiology | 2015‬‬ ‫הצטרפתי לפרויקט ח\"ץ בשנת ‪ 2012‬ומאז חנכתי תשעה סטודנטים‬ ‫מנחה‪ :‬פרופ' אלדד קטורזה‬ ‫לרפואה ושני תלמידי תיכון‪ ,‬כולם מבריקים בצורה יוצאת דופן ומעוררת‬ ‫הערכה רבה‪ .‬במהלך תקופה זו פרסמנו שמונה מאמרים מדעיים במיטב‬ ‫דימות העובר‪ ,‬יילוד וגינקולוגיה‬ ‫מנהל פרויקט ח\"ץ‬ ‫העיתונים בתחום המחקר בו אני עוסק‪.‬‬ ‫מנהל מכון גרטנר‬ ‫הפרויקט תרם לי רבות מבחינה מקצועית ואישית ומהווה כבוד וזכות‬ ‫‪[email protected]‬‬ ‫גדולה להשתייך אליו‪ .‬היכולת לעצב ולהשפיע על דור העתיד של מערכת‬ ‫הבריאות בארץ ובעולם היא לא דבר מובן מאליו‪.‬‬ ‫יישר כח לכל העוסקים והמשתתפים במיזם חשוב וייחודי זה‪.‬‬ ‫רועי בר‬ ‫אונ' תל אביב‬ ‫השתתף כסטודנט בפרויקט ח“ץ‬ ‫בין השנים ‪2012-2014‬‬ ‫‪[email protected]‬‬ ‫‪209‬‬

ORIGINAL RESEARCH PEDIATRICS Normal Fetal Posterior Fossa in MR Imaging: New Biometric Data and Possible Clinical Significance R. Ber, O. Bar-Yosef, C. Hoffmann, D. Shashar, R. Achiron, and E. Katorza ABSTRACT BACKGROUND AND PURPOSE: Posterior fossa malformations are a common finding in prenatal diagnosis. The objectives of this study are to re-evaluate existing normal MR imaging biometric data of the fetal posterior fossa, suggest and evaluate new parameters, and demonstrate the possible clinical applications of these data. MATERIALS AND METHODS: This was a retrospective review of 215 fetal MR imaging examinations with normal findings and 5 examinations of fetuses with a suspected pathologic posterior fossa. Six previously reported parameters and 8 new parameters were measured. Three new parameter ratios were calculated. Interobserver agreement was calculated by using the intraclass correlation coefficient. RESULTS: For measuring each structure, 151–211 MR imaging examinations were selected, resulting in a normal biometry curve according to gestational age for each parameter. Analysis of the ratio parameters showed that vermian lobe ratio and cerebellar hemisphere ratio remain constant with gestational age and that the vermis-to-cisterna magna ratio varies with gestational age. Measurements of the 5 pathologic fetuses are presented on the normal curves. Interobserver agreement was excellent, with the intraclass correlation coefficients of most parameters above 0.9 and only 2 parameters below 0.8. CONCLUSIONS: The biometry curves derived from new and existing biometric data and presented in this study may expand and deepen the biometry we use today, while keeping it simple and repeatable. By applying these extensive biometric data on suspected abnormal cases, diagnoses may be confirmed, better classified, or completely altered. ABBREVIATIONS: ICC ϭ intraclass correlation coefficient; CHR ϭ cerebellar hemisphere ratio; CMS ϭ cisterna magna cross-sectional area; PF ϭ posterior fossa; TCD ϭ transcerebellar diameter; VCMR ϭ vermis-to-cisterna magna ratio; VLR ϭ vermian lobe ratio; VP ϭ vermian perimeter; VS ϭ vermian cross- sectional area The posterior cranial fossa is located between the foramen mag- rhombencephalon (pons and cerebellar hemispheres), and the num, which forms its caudal boundary, and the tentorium myelencephalon part of the rhombencephalon (medulla oblon- cerebelli, which form its cephalad boundary. It includes the 3 gata), starting at the fourth gestational week. Normally, by the parts of the brain stem: medulla oblongata, pons, and midbrain; 18th gestational week, the PF consists of a developed vermis and the cerebellum with its vermis; and the fluid-filled spaces: the cerebellum, a developed pons, and a fourth ventricle fully covered fourth ventricle and cisterna magna. The structures of the poste- by the caudal part of the vermis, and the fluid-filled spaces are rior fossa (PF) develop from the mesencephalon of the neural connected by the foramina of Luschka and Magendie.1,2 tube (midbrain and vermis), the metencephalon part of the During the development of the structures and fluid-filled Received August 4, 2014; accepted after revision September 22. spaces of the PF, a wide spectrum of malformations of these struc- From the Departments of Obstetrics and Gynecology (R.B., D.S., R.A., E.K.), Pediat- tures is often observed. The variety of these malformations, iso- ric Neurology (O.B.-Y.), and Diagnostic Imaging (C.H.), Chaim Sheba Medical Cen- lated or part of a syndrome, and of their outcomes makes it diffi- ter, Tel Hashomer, Tel-Aviv University, Tel-Aviv, Israel. cult to classify the different pathologies.3,4 During the past decade, Please address correspondence to Roee Ber, Department of Obstetrics and Gyne- attempts to classify the PF malformations have been made, to help cology, Chaim Sheba Medical Center, Tel Hashomer, Tel-Aviv University, Tel-Aviv, physicians and radiologists diagnose and give an accurate prog- Israel; e-mail: [email protected] nosis for fetuses with a pathologic PF.5-7 Indicates article with supplemental on-line tables. The different classifications and the possible diagnoses are Indicates article with supplemental on-line photos. mainly on the basis of the morphology of the fetal PF and its http://dx.doi.org/10.3174/ajnr.A4258 biometric data. Formerly, normal biometric data were taken AJNR Am J Neuroradiol 36:795– 802 Apr 2015 www.ajnr.org 795 210

FIG 1. New parameters measured. A, Sagittal section of the PF: 1) vermian anterior lobe uating the vermis and better contrast cross-sectional area, 2) vermian posterior lobe cross-sectional area, 3) pontine height, 4) resolution, which enable evaluation of pontine cross-sectional area, 5) cisterna magna cross-sectional area, 6) brain stem cross the brain stem.13,14 Therefore, during sectional area. B, Axial section of the PF: 1) cerebellar cross-sectional area and perimeter, and the past 2 decades, MR imaging has be- 2) cerebellar hemisphere cross-sectional areas. come an important tool to prenatally evaluate the morphology of the PF, and many studies have been published to supply valid MR imaging biometric data.15-17 However, because using MR imaging prenatally is still not a common procedure and is usually performed on suspected abnormal fetuses, re-evaluation is still needed. In addition, the high reso- lution of MR images makes it possible to measure structures that could not be mea- sured before. In this study, we re-evaluated exist- ing normal MR imaging biometric data of PF structures in a large cohort. In addition, we suggest new biometric data, that, to our knowledge, were not measured in previous studies, to help correctly diagnose suspected patho- logic fetuses. We demonstrate the po- tential clinical use of these data by ret- rospectively evaluating 5 different suspected pathologic cases. MATERIALS AND METHODS Population We performed a retrospective review of 215 fetal MR imaging examinations in the Chaim Sheba Medical Center between 2007 and 2013. The included examina- tions were selected according to the fol- lowing criteria: 25th–39th gestational week (the distribution of the number of fetuses examined by gestational week is presented in On-line Fig 1), no PF find- ings, and mild-to-no cerebral findings. Examinations of fetuses with isolated ex- tracranial anomalies or maternal cyto- megalovirus infection with no intracranial anomalies were also included. Eighty per- cent of the fetuses had no abnormal find- ings, and 20% had a mild lateral ventricu- lar asymmetry or ventriculomegaly. The list of examination indications and find- ings is presented in On-line Table 1. For each structure measured, only satisfactory images in terms of quality and alignment FIG 2. Percentile curves for new parameters. A, Vermian anterior lobe cross-sectional area. B, were selected to be measured. Vermian posterior lobe cross-sectional area. In addition, 5 fetuses with a sus- pected pathologic PF were selected for re-evaluation according to the new ref- mainly from studies of PF biometry in ultrasonographic imag- erence data generated in this study. The same structures were ing.8-12 However, MR imaging has advantages over sonogra- measured for these fetuses with the same procedure performed phy when assessing fetal PF, such as better accuracy when eval- for healthy fetuses and detailed below. 796 Ber Apr 2015 www.ajnr.org 211

FIG 2. Continued. C, Pontine height. D, Pontine cross-sectional area. dom fetuses were remeasured by an- other operator (E.K.) to evaluate MR Imaging Technique interobserver agreement for each In our institution, we perform fetal brain MR imaging by using a structure. For each examination, 9 pa- 1.5T system (Optima; GE Healthcare, Milwaukee, Wisconsin). rameters were measured in the mid- Single-shot fast spin-echo T2-weighted sequences in 3 orthogonal sagittal section and 5, in the axial sec- planes were performed by using a half Fourier technique (NEX ϭ tion. Axial section parameters were 0.53) with the following parameters: section thickness, 3– 4 mm; measured slightly above the base of no gap; flexible coil (8-channel cardiac coil); matrix, 320/224; TE, skull in the plane of the following 90 ms; and TR, 1298 ms. The FOV was determined by the size of landmarks: the fastigium of the fourth the fetal head: 24 cm for the smaller fetuses and up to 30 cm for the ventricle, pons, anterior part of the larger fetuses. T1 fast-spoiled gradient-echo sequences were per- temporal lobes, and eyeballs, with a formed only in the axial plane with a larger FOV (400 mm); sec- symmetric presentation of the tempo- tion thickness, 4 mm; gap, 0.5 mm; TR, 160 ms; and TE, 2.3 ms. ral lobes and eyeballs. Previously re- The in-plane resolution of the T1 fast-spoiled gradient-recalled ported parameters measured in the images was the following: sagittal—matrix, 256 ϫ 160; FOV, midsagittal section included the an- 30 ϫ 30; voxel, 1.17 ϫ 1.875 mm; and coronal: matrix, 256 ϫ 160; teroposterior diameter of the vermis, FOV, 36 ϫ 36; voxel, 1.4 ϫ 2.25 mm. vermian height, vermian perimeter (VP), vermian cross-sectional area MR imaging was followed by a DWI sequence performed with (VS), and pontine anteroposterior di- a 40-cm FOV and b-values of 0 and 1000 or 700 ms. The ADC ameter. Previously reported parame- calculation map was added.18 ters measured in the axial section included transcerebellar diameter Measurements (TCD). New parameters measured in the All measurements were performed manually by a single oper- midsagittal section are presented in Fig 1A ator (R.B.) on a PACS reading workstation. Twenty-five ran- and included the vermian anterior lobe cross-sectional area, vermian posterior lobe cross-sectional area, pontine height, pontine cross-sectional area, brain stem cross-sectional area, and cisterna magna cross-sectional area (CMS). New parame- ters measured in the axial section are presented in Fig 1B and included the cere- bellar perimeter and cerebellar cross-sec- tional area. We calculated the following additional parameter ratios: the ratio between the cross-sectional areas of the vermian lobes (VLR), the ratio between the VS and CMS (VCMR), and the ratio between the cross-sectional areas of the cerebellar hemi- spheres (CHR). Previously reported biometric parameters (an- teroposterior diameter of the vermis, vermian height, VS, VP, pontine anteroposterior diameter, TCD) were measured ac- cording to common methodology.15,19 New parameters intro- duced in this study were measured as follows. Sagittal Section Measurements. Vermian anterior lobe cross- sectional area and vermian posterior lobe cross-sectional area were measured as the cross-sectional area of the corresponding lobe of the vermis, with a separation line between the fourth ventricle fastigium and the vertex of the primary fissure of the vermis (Fig 1A). The posterolateral fissure, which separates the vermis from the nodule, is difficult to recognize in most im- ages; therefore, the nodule of the vermis was included in the vermian posterior lobe cross-sectional area (Fig 1A). Pontine height was measured as the height between the upper notch created between the pons and the cerebral peduncle and the lower notch created by the pontomedullary angle (Fig 1A). The pontine cross-sectional area was measured as the area be- AJNR Am J Neuroradiol 36:795– 802 Apr 2015 www.ajnr.org 797 212

our study, the model for centile q at gestational age t was Cq ϭ ␮t ϩ ␴tZq, where ␮t and ␴t were the mean and SD at age t, measured in days, and Zq was the q centile of the standard normal distribution. The functions ␮t and ␴t were estimated and smoothed by using the Rigby and Stasinopoulos algo- rithm20 with a cubic spline smoothing. The normality assumption was slightly in- adequate, but the resulting curves were al- most identical to those achieved by assum- ing the Box-Cox t distribution (with 4 parameters) recommended.21 In addi- tion, we found the skewness and kurtosis parameters of the Box-Cox t distribution to be nonsignificant for all response variables; this finding supports our decision to simply use the normal distribution without any transformation. For the ratio variables (VCMR, VLR, CHR), we examined the hypothesis ␮t ϭ ␮, to assess the independence of the ra- tios with gestational age. The hypothesis was tested by using the Generalized Ad- ditive Models for Location, Scale and Shape. Intraclass correlation coefficient (ICC) and limit of agreement were used to study the reliability of mea- surements across measurers, and 25 FIG 2. Continued. E, Cisterna magna cross-sectional area. F, Brain stem cross-sectional area. subjects were measured by 2 measur- ers for this purpose. Results were de- tween 2 lines stretched from the pontopeduncular notch and the fined as poor for ICC Ͻ 0.6, satisfactory for 0.6 Ͻ ICC Ͻ 0.8, pontomedullary notch described above to the posterior boundary good for 0.8 Ͻ ICC Ͻ 0.9, and excellent for ICC Ͼ 0.9. of the brain stem and orthogonal to its axis (Fig 1A). CMS was measured as the area whose boundaries were the Ethics Approval tentorium cerebelli, the posterior boundary of the brain stem, and The research was approved by the hospital research ethics board. the foramen magnum. The vermis, the fourth ventricle, and the cisterna magna were included in the CMS (Fig 1A). RESULTS Brain stem cross-sectional area was measured as the cross- Normal Biometric Reference Data Two hundred fifteen MR imaging examinations were selected sectional area of the medulla, the pons, and the midbrain with the tectum of the midbrain included (Fig 1A). for measurement, of which 151–211 images were selected as Axial Section Measurements. The cerebellar perimeter and cere- adequate for measuring in terms of quality and alignment, for bellar cross-sectional area were measured as the perimeter and area, each structure. The number of images per structure is pre- respectively, encircling the cerebellum and the pons (Fig 1B). sented in On-line Table 2. Normal percentile curves of biomet- ric reference data previously reported (anteroposterior diam- Cross-sectional areas of the cerebellar hemispheres were mea- eter of the vermis, vermian height, VS, VP, pontine sured as the area of right and left hemispheres of the cerebellum anteroposterior diameter, TCD) are presented in On-line Fig alone, excluding the vermis (Fig 1B). 2. Normal percentile curves of new biometric reference data Statistical Analysis (vermian anterior lobe cross-sectional area, vermian posterior All statistical analysis was performed by using R, Version 3.0.1 lobe cross-sectional area, pontine height, pontine cross-sec- (R statistical computing software; http://www.r-project.org). tional area, CMS, brain stem cross-sectional area, cerebellar The reference intervals were estimated by using the General- perimeter, cerebellar cross-sectional area) are presented in Fig ized Additive Models for Location, Scale, and Shape model,20 2. Normal percentiles for each parameter by gestational age are the suggested method of the World Health Organization.21 In presented in On-line Tables 3–16. 798 Ber Apr 2015 www.ajnr.org 213

measurements of images are presented on the normal curves in On-line Fig 2 and Fig 2 and are labeled according to their case number in the Table. Normal Parameter Ratios For VCMR, we obtained a highly signif- icant result (P Ͻ 10Ϫ10), implying that VCMR is not constant with gestational age. The normal percentile curves for VCMR are presented in On-line Fig 3, and the normal percentiles for VCMR are presented in On-line Table 17. For VLR and CHR, we obtained P ϭ .09 and P ϭ .59, respectively, and concluded that these ratios are constant with gestational age. Normal percentiles for VLR and CHR are presented in On-line Table 18. Interobserver Agreement Agreement between the 2 operators for each structure showed excellent correla- tion (ICC Ͼ 0.9) between measurers in 8 parameters, good correlation (0.8 Ͻ ICC Ͻ 0.9) in 4 parameters, and satisfac- tory correlation in only 2 parameters, VP and pontine height. The results are presented in On-line Table 19. DISCUSSION Malformations of the posterior fossa are a common finding in prenatal diagnosis FIG 2. Continued. G, Cerebellar perimeter. H, Cerebellar cross-sectional area. and include a wide variety of pathologies and a wide spectrum of prognoses.1,11,22 Abnormal measurements of pathologic cases Therefore, the exact diagnosis and accu- rate prognosis given to the soon-to-be Gestational Indication for MRI parents are crucial for the their under- Case Age (weeks) Abnormal Measurements standing of the consequences of these malformations. The establishment of a A 31.0 Cerebellar asymmetry CHR, TCD, CS, and CP below diagnosis of PF malformation is based on several parameters, including anat- 3rd percentile omy, morphology, and biometry.7 In- sufficient biometry and reliance on ex- B 31.6 Enlarged cisterna magna No abnormal measurements aminers’ subjective assessments may lead to a wrong diagnosis and wrong C 32.0 Suspected abnormal vermis VS, VPLS, and VCMR below prognosis, including under- or overdi- agnosis of different pathologies.23,24 3rd percentile; CMS and VLR Existing 2D MR imaging biometric above 97th percentile data of the PF are limited to only a few D 34.0 Suspected abnormal vermis APDV, VH, VS, VP, VALS, and VPLS below 3rd percentile E 32.0 Low TCD measurements TCD, CS, and CP below and suspected abnormal 3rd percentile brain stem Note:—APDV indicates anteroposterior diameter of the vermis; VH, vermian height; VALS, vermian anterior lobe cross-sectional area; CP, cerebellar perimeter; CS, cerebellar cross-sectional area; VPLS, vermian posterior lobe cross- sectional area. Pathologic Cases Biometry parameters, which reduce the ability to We obtained abnormal measurements for cases A, C, D, and E, analyze dynamic components such as the PF structures. Thus, according to new and previously reported biometric data. Case B the physician evaluating the fetus must rely on his or her subjec- had no abnormal measurements according to the parameters tive evaluation with limited objective tools. Furthermore, some measured in this study. A list of the pathologic cases, their indica- previously reported studies showed no benefit in diagnosing PF tion for MR imaging examination, and their abnormal biometry malformations by using MR imaging compared with ultra- findings (measurements exceeding 2 SDs) is presented in the Ta- sound.4,22,25 However, the increasing use of fetal MR imaging ble. The images of these fetuses are presented in Fig 3, and the examinations after ultrasound screening to diagnose cerebral pa- AJNR Am J Neuroradiol 36:795– 802 Apr 2015 www.ajnr.org 799 214

tool for diagnosing PF malformations. We performed measurements on a large cohort of 215 fetuses from the 25th to 39th gestational week. We measured 6 previously reported parameters15,19 (anteroposterior diameter of the vermis, vermian height, VS, VP, pontine antero- posterior diameter, TCD) that are rou- tinely used as biometric data. These pa- rameters, together with morphologic analysis by the physician, support the di- agnosis of PF malformations. To these existing data, we added 8 new biometric parameters and 3 new ratio parameters. We provided normal curves of these pa- rameters and third, 15th, 50th, 85th, and 97th percentiles. We also showed the reproducibility of these measurements by evaluating interobserver agreement. Only 2 parameters, VP and pontine height, showed a satisfactory agreement. This could be explained by the difficulty in recognizing small fissures of the ver- mis in the case of VP and the difficulty in determining the location of the pon- tomedullary angle in the case of pontine height. The rest of the parameters mea- sured showed good-to-excellent inter- observer agreement. These expanded biometric data may replace the subjec- tive morphologic assessment and allow further objective investigation of the pathologies. Case A is an example of the possible clinical significance of the new parame- ters. Cerebellar asymmetry is a pathology diagnosed by the physician by morpho- logically evaluating the fetal cerebellum, along with measuring a small TCD. The FIG 3. Pathologic cases indicated for MR imaging examinations. A, Cerebellar asymmetry. B, cerebellar hemisphere ratio parameter, in- Enlarged cisterna magna. C, Suspected abnormal vermis. D, Suspected abnormal vermis. E, Low troduced in this study, may help the phy- TCD measurements in axial (left) and saggital (right) planes. sician determine whether the asymmetry between lobes is within normal limits. In thologies enables us to expand our biometric data to finer struc- this case, the CHR was measured as 1.52, which exceeds normal lim- tures such as the vermian lobes and the brain stem. The vermian its and validates the subjective diagnosis of cerebellar asymmetry. By primary fissure is difficult to recognize by using ultrasound, mak- these data, we turn subjective analysis into an objective biometry- ing it difficult to differentiate between the vermian lobes. The based analysis. brain stem is considered difficult to depict by using ultrasound “Mega cisterna magna” is historically defined as a cisterna techniques, and only limited biometric data are available.10 MR magna diameter exceeding 10 mm.30 Previous studies have re- imaging allows better evaluation of these structures and possible ported this definition to be inaccurate, and it has been shown to biometric assessment that might reinforce the role of MR imaging vary with gestational age.31,32 The cisterna magna is a fluid-filled after ultrasound screening. Recent studies suggest a challenging space that is normally continuous with the subarachnoid space of approach to this issue by developing the technology to expand to the entire PF, between the foramen magnum and the tentorium 3D MR imaging biometry.26-29 However, this approach has not cerebelli. We measured the cross-sectional area of the entire fluid- matured and is not yet in clinical use. filled space in the midsagittal section (CMS) and the ratio between In this study, we suggest that a more comprehensive yet tech- the vermian cross-sectional area and the CMS. As reported and seen nologically simple 2D biometry, including biometric relations be- in Figs 2, both CMS and VCMR change according to gestational age tween structures, can be a more thorough and accurate objective but at different rates due to the change in VS. Case B was referred for 800 Ber Apr 2015 www.ajnr.org 215

MR imaging due to an enlarged cisterna magna on ultrasound exam- This effect was previously reported to be significant statistically ination; the cisterna magna was measured as Ͼ10 mm on MR imag- but insignificant clinically.17 ing. However, the new parameters, CMS and VCMR, were normal for this case. We believe that these curves may suggest a different CONCLUSIONS approach for diagnosing mega cisterna magna, both isolated and as Diagnosis of fetal pathologies of the PF are based mainly on bi- part of a more complex PF malformation. Perhaps by redefining the ometry and morphology of the different structures. The expand- criteria for mega cisterna magna according to the new parameters, ing usage of MR imaging in prenatal screening enables us to ex- CMS and VCMR, we may avoid overdiagnosis of mega cisterna pand the biometry we use in sonography screening. In this study, magna. we presented comprehensive normative data, including the biom- etry of previously reported and new parameters and parameter Cases C and D were both referred for MR imaging examina- ratios, in MR imaging, while keeping them simple and repeatable. tion due to a suspected abnormal vermis on ultrasound. As seen in We suggest that applying these new data may help further classify Fig 3, both cases present a rotated and small vermis. Indeed, VS posterior fossa malformations, confirm borderline diagnoses, and parameters for the 2 cases are below the third percentile. Never- avoid over- and underdiagnoses. theless, when we applied the new parameter measurements, a dif- ferentiation between those cases could be achieved. Case C pre- REFERENCES sented with an abnormal ratio between the vermian lobes, VLR above the 97th percentile, with a small posterior lobe. This finding 1. Adamsbaum C, Moutard ML, Andre C, et al. 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Mitochondrial induction as a potential radio-sensitizer in lung cancer cells - a short report Cellular oncology | 2015 ‫ פרופ׳ ניר פלד‬:‫מנחה‬ ‫אונקולוגיה‬ [email protected] ‫רונן שביט‬ ‫אונ' תל אביב‬ ‫השתתף כסטודנט בפרויקט ח״ץ‬ 2012-2015 ‫בין השנים‬ [email protected] 219

Cell Oncol. (2015) 38:247–252 DOI 10.1007/s13402-014-0212-6 ORIGINAL PAPER Mitochondrial induction as a potential radio-sensitizer in lung cancer cells - a short report Ronen Shavit & Maya Ilouze & Tali Feinberg & Yaacov Richard Lawrence & Yossi Tzur & Nir Peled Accepted: 10 December 2014 / Published online: 7 January 2015 (35 to 17 %, p=0.28) respectively, compared to radiation # International Society for Cellular Oncology 2015 alone. Conclusion Mitochondrial induction by DCA may serve as a Abstract radio-sensitizer in non-small cell lung cancer. Introduction Lung cancer is the leading cause of cancer death. Radiation therapy plays a key role in its treatment. Ionizing Keywords NSCLC . DCA . Mitochondria . Radiation . radiation induces cell death through chromosomal aberrations, Radio-sensitizer . Warburg effect which trigger mitotic catastrophe and apoptosis. However, many lung cancer patients show resistance to radiation. 1 Introduction Dichloroacetate (DCA) is a small molecule that can promote mitochondrial activation by increasing the influx of pyruvate. Lung cancer is the leading cause of cancer-related deaths in Here, we tested whether DCA may increase the sensitivity of the United States with an overall 5-year survival rate for all non-small cell lung cancer (NSCLC) cells to radiation through stages of ~17 % [1–4]. Radiation therapy (RT) plays an this mechanism. important role in the clinical management of lung cancer Methods Two representative NSCLC cell lines (A549 and patients, particularly those with stage IIIB disease who are H1299) were tested for their sensitivity to radiation with and candidates for definitive chemo-radiotherapy. In addition, RT without pre-exposure to DCA. The treatment efficacy was may be applied as a neo-adjuvant or adjuvant therapy in stage evaluated using a clonogenic survival assay. An extracellular IIIA, or in an ablative manner when stereotactic body radio- flux analyzer was used to assess the effect of DCA on cellular therapy (SBRT) is applied. Radiation induced pneumonitis oxygen consumption as a surrogate marker for mitochondrial (RIP) is the limiting factor when treating patients with RT. activity. In order to minimize RIP, oncologists aim to keep the V20 Results We found that DCA increases the oxygen consump- (i.e., the percentage of lung volume receiving a radiation dose tion rate in both A549 and H1299 cells by 60 % (p=0.0037) of ≥20 Gy) below 22 % [5]. Radio-sensitizers may increase and 20 % (p=0.0039), respectively. Pre-exposure to DCA one the cytotoxic efficacy of radiation, thereby potentially improv- hour before radiation increased the cytotoxic death rate 4-fold ing cure rates without increasing the V20. in A549 cells (55 to 13 %, p=0.004) and 2-fold in H1299 cells RT kills cells by causing DNA double-strand breaks. R. Shavit : M. Ilouze : T. Feinberg : Y. Tzur : N. Peled (*) Unrepaired DNA breaks result in chromosomal aberrations which, in turn, lead to “mitotic catastrophe” - a mode of cell Thoracic Cancer Research and Detection Center, Sheba Medical death that results from the premature or inappropriate entry of Center Tel Hashomer, Ramat-Gan 52621, POB 244, Israel cells into mitosis [6, 7]. Furthermore, radiation may directly e-mail: [email protected] affect cell membranes and organelles. Although these latter URL: http://medicine.mytau.org/peled/ changes are as yet poorly understood, they may lead to chang- es in signal transduction, gene expression, protein stability, Y. R. Lawrence cellular redox states and cell cycle regulation, all of which Center for Translational Research in Radiation Oncology, Sheba may lead to apoptosis [8]. Moreover, radiation may induce Medical Center, Ramat-Gan, Israel M. Ilouze : N. Peled Thoracic Cancer Service, Davidoff Cancer Center, Rabin Medical Center, Petach Tikva, Israel 220

248 R. Shavit et al. mitochondrial reactive oxygen species (ROS) production, ac- promoting its oxidation via mitochondrial activity over gly- companied by up-regulation of mitochondrial electron trans- colysis in various cancer types (depicted in Fig. 1) [13, 18]. port chain functions, thereby increasing mitochondrial mem- An increase in mitochondrial activity causes an increase in brane potential, mitochondrial respiration and mitochondrial both the amount and the extent of release of free radicals ATP production [9, 10]. However, genetic alterations limit the (ROS) in the cell, leading to an increase in cellular apoptotic capacity of cancer cells to undergo apoptosis, suggesting that activity [13]. hypoxic tumor microenvironments may provide a selective pressure towards an apoptosis-resistant cancer phenotype and, Here, we investigated the potential radio-sensitizing effect as a result, resistance to RT [11]. Because of this RT resistant of DCA in NSCLC cells. Our results indicate that mitochon- phenotype and the significant side effects caused by RT itself, drial activation by DCA increases the sensitivity to radiation it is of a crucial importance to explore ways to radio-sensitize in both A549 and H1299 NSCLC-derived cells. lung cancer cells in order to decrease radiation doses and to improve responses to therapy. 2 Materials and methods Many tumor cells exhibit elevated levels of glucose uptake 2.1 Cell cultures and reduced levels of oxidative phosphorylation. This para- doxical hyper-glycolysis and lack of mitochondrial activity in Two non-small cell lung cancer (NSCLC) cell lines were used the presence of oxygen is known as Warburg effect (depicted in this study: A549, derived from a human adenocarcinoma in Fig. 1) [12]. The unique metabolism of most solid tumors and H1299, derived from a human large cell carcinoma. Both stems from remodeling mitochondrial functions to produce a cell lines were purchased from the ATCC. The cells were glycolytic phenotype and a strong resistance to apoptosis [13]. cultured in RPMI-1640 medium supplemented with 10 % There is a growing body of evidence indicating that mitochon- fetal bovine serum (FBS), 1 % penicillin/streptomycin and dria may be the primary targets for cancer therapeutics 1 % glutamate at 37 °C in a 5 % CO2 humidified atmosphere. [14–17]. Cancer-specific remodeling can be reversed by a small molecule named dichloroacetate (DCA) [18], which 2.2 Treatment conditions inhibits pyruvate dehydrogenase kinase (PDK), thereby in- creasing the influx of pyruvate into the mitochondria and Cancer cell metabolism Effect of DCA Dichloroacetate (DCA, Sigma 34795) was added to culture Glucose media for 1 h prior to radiation at 10 and 20 mM in case of Glucose H1299 cells and 40 and 60 mM in case of A549 cells, Pyruvate DCA according to the respective IC50 values. Cells were radiated PDH Pyruvate with a Kimtron Polaris irradiator at a dose rate of 0.9 Gy/min at room temperature. PDK 2.3 Clonogenic survival assay PDH PDK ROS The clonogenic survival assay that we used has been previ- ROS ously described [19]. Briefly, cells were detached with trypsin, suspended in complete culture media, counted and seeded in Radiation ROS ROS ROS 3 ml dishes at a density of 200 cells per dish, and then left to ROS ROS attach and stabilize overnight. The next day, test groups were RORS adiation treated with DCA (or control) 1 h before radiation, at the ROS indicated concentrations. Cells were radiated to doses of 0, 2, 4 and 6 Gy. Following incubation for 48 h, the culture Cell death decreased Cell death increased media were removed and replaced with fresh culture media without DCA (all groups). Cells were incubated for another DCA- Dichloroacetate. ROS- Reactive oxygen species .PDH- Pyruvate dehydrogenase. 10 days to allow colony formation. Next, colonies were fixed and stained with 0.5 % crystal violet in 96 % ethanol. Colonies PDK- Pyruvate dehydrogenase kinase - Inhibition. – Propagation. containing at least 50 viable cells were counted (Fig. 2). Each experiment was performed in duplicate and the mean and Fig. 1 Schematic representation of the proposed effect of DCA on cancer standard deviation were calculated from 3 independent cells exposed to radiation. The left panel depicts the Warburg effect: even experiments. in the presence of oxygen, cancer cell metabolism relies on glycolysis. This is accomplished by the intracellular expression of PDK which, in turn, inhibits the action of PDH. The result is inhibition of the influx of pyruvate to mitochondria and, thus, mitochondrial activity. In spite of DNA damage caused by radiation, cancer cells are unable to undergo efficient apoptosis since apoptotic modulators like reactive oxygen species (ROS) remain in the mitochondria. The right panel depicts the effect of DCA: DCA abolishes the inhibition of PDH by inhibiting PDK. Consequently, DCA increases the influx of pyruvate into the mitochondria and, thus, mitochondrial activity. In addition to DNA damage due to radiation, apoptotic modulators are released from the mitochondria and, hence, cancer cells undergo a higher rate of apoptosis 221

Mitochondrial induction – a radio-sensitizer in lung cancer cells 249 Flow chart 2.4 Synergy evaluation Day 1 Cell seeding A549 cells H1299 cells In order to evaluate whether the treatment schedules show synergistic effects, plots of averages with standard deviations Day 2 DCA treatment-1 hour before of survival rates relative to radiation doses were made. Sur- irradiation vival percentages were calculated, both for groups treated and untreated with DCA, relative to those of unirradiated cells: Radiation treatment 0 Gy 2 Gy 4 Gy 6 Gy survival ¼ Plating efficiency of irradiatied cells � 100%ð1Þ plating efficiency of unirradiated cells Incubation for 48 hours Day 4 DCA wash where: Colony fixation Incubation for 10 days number of colonies formed number of cells seeded Day 14 plating efficiency ¼ � 100% ð2Þ Analysis Specifically, colony formation of both DCA-treated and DCA-untreated groups without radiation treatment were Fig. 2 Flow chart of the clonogenic survival assay b a cd * * Fig. 3 Clonogenic survival of NSCLC cells treated with increasing DCA with radiation doses of 2, 4, 6 Gy alone and combined with 40 and 60 mM consentrations. a A549 cells treated with 20, 40, 60 and 80 mM DCA. b DCA. d H1299 cells treated with radiation doses of 2, 4, 6 Gy alone and H1299 cells treated with 10, 20, 30 and 50 mM DCA. c A549 cells treated combined with 10 and 20 mM DCA. *p<0.05 compared to control 222

250 R. Shavit et al. a 2.6 Statistical analysis Statistical significance was evaluated using Student’s t-test. Synergy was evaluated by a two-way ANOVA [20]. A value of p<0.05 was considered significant. 3 Results 3.1 DCA radio-sensitizes NSCLC cells b Dichloroacetate (DCA) is known to increase mitochondrial activity [10, 15] and has previously been shown to act as a Fig. 4 Oxygen consumption rate (OCR) in A549 and H1299 cells. DCA radio-sensitizer in several cancer cell lines derived from pros- was added after ~16 min. a 20 mM DCA added to H1299 cells. b 40 mM tate, colorectal and brain tumors [21, 22]. Here, its effect on DCA added to A549 cells. OCR changes were measured relative to basal two non-small cell lung cancer (NSCLC)-derived cell lines states. DCA increased the oxygen consumption rate in A549 and H1299 was investigated, i.e., A549 (adenocarcinoma) and H1299 cells by 60 % (p=0.0037) and 20 % (p=0.0039), respectively (large cell carcinoma). The concentrations of DCA used for each cell line were in accordance with the respective IC50 normalized to 100 %. Synergistic effects were determined by a values (A549: 63 mM and H1299: 36 mM; Fig. 3a and b). two-way ANOVA (p value <0.05) [20]. We found that pre-radiation exposure with DCA increased 2.5 Oxygen consumption assay cell death in both A549 and H1299 cell samples. The maximal effect in A549 cells was observed at 4 Gy, where DCA Oxygen consumption was used as a surrogate marker for treatment (40 and 60 mM) decreased cell survival from ~55 mitochondrial activity. Measurements were carried out using to 40 % (p=0.27) and 13 % (p=0.004), respectively (Fig. 3c). an XF24 extracellular flux analyzer (Seahorse Biosciences). In H1299 cells, the maximal effect was reached at 6 Gy. At This device uses fluorescence-based optical sensors and cus- this radiation dose, DCA treatment (10 and 20 mM) decreased tom multi-well plates to perform repeated oxygen consump- cell survival from 35 % (radiation only) to 25 and 17 %, tion measurements of intact cells growing as monolayers. respectively (not statistically significant; Fig. 3d). Pre- H1299 and A549 cells were seeded in Seahorse XF24 cell exposure of A549 cells to DCA (60 mM) increased the cyto- culture plates at 10,000 and 20,000 cells per well, respectively, toxic effect of 4 Gy radiation by 4-fold compared to radiation in growth medium. Cells were incubated for 48 h at 37 °C in alone. A two-way ANOVA analysis indicated a synergistic 5 % CO2. Next, cells were washed and transferred to XF assay effect only in A549 cells. medium/PBS, and incubated for 60 min at 37 °C without CO2 before starting the experiment. After establishing baseline 3.2 DCA increases oxygen consumption in both A549 oxygen consumption rates, 20 and 40 mM DCA were added and H1299 cells to H1299 and A549 cells, respectively. Oxygen consumption measurements were continued for 25 min. Data were acquired An enhanced cellular oxygen consumption rate (OCR) is an from at least three replicate plates per cell line. Results are indication of increased mitochondrial activity [23]. We found represented as the percent of the baseline respiration rate. that DCA increases the OCR in both A549 and H1299 cells by 60 % (p=0.0037) and 20 %, (p=0.0039), respectively, com- pared to the basal OCR (Fig. 4). 4 Discussion By testing the sensitivity to radiation with and without pre- exposure to dichloroacetate (DCA), we found that mitochon- drial activation by DCA can radio-sensitize two distinct non- small cell lung cancer (NSCLC)-derived cell lines (i.e., A549 and H1299). In this proof of concept study, a wide range of 223

Mitochondrial induction – a radio-sensitizer in lung cancer cells 251 DCA concentrations was used, aiming to tailor the dose to radicals and apoptosis by DCA, is the leading cause of DCA’s each cell’s IC50 value. The synergistic effect of DCA was potential in radio-sensitizing NSCLC cells. statistically significant in A549 cells, while only approaching significance in H1299 cells. Possibly, an even more robust In summary, we show that in vitro mitochondrial induction effect can be obtained with higher DCA doses. We also by DCA significantly radio-sensitizes A549 NSCLC cells in a showed that DCA increases oxygen consumption in both synergistic manner. This observation warrants further assess- A549 and H1299 cells, which is a surrogate marker for in- ment in an in vivo setting. creased mitochondrial activity. Acknowledgments The authors thank Dr. Shoshana Paglin (Sheba Ionizing radiation up-regulates the mitochondrial electron Medical Center) for guidance and fruitful discussions regarding this transport chain function, and increases mitochondrial mem- work. brane potential and ATP production [9]. Mitochondrial hyper- activation by DCA further increases the amount and the extent This work was performed in partial fulfillment of the MD thesis of the released free radicals (ROS) by the mitochondria itself requirements of the Sackler Faculty of Medicine, Tel Aviv University [13]. These factors may explain the synergistic effect observed and was supported by Sheba Medical Center. (Mr. Ronen Shavit) by pre-exposing the cells to DCA before starting radiation. Conflict of interest The authors declare that they have no conflict of Atkinson et al. 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‫‪Reduced expression and growth inhibitory activity of the‬‬ ‫‪aging suppressor klotho in epithelial‬‬ ‫‪Cancer Letters | 2015‬‬ ‫שמחה מאוד להשתתף כמנחה בפרויקט ח״ץ המעולה שגדל ומשתבח כל‬ ‫הזמן‪ .‬המפגש עם הסטודנטים נותן הרבה תקווה ואופטימיות לגבי עתיד‬ ‫הרפואה‪.‬‬ ‫מנחה‪ :‬דר' רות חונא‪-‬בר‪-‬און‬ ‫עיניים‬ ‫‪[email protected]‬‬ ‫עומר שוורצמן‬ ‫אונ' תל אביב‬ ‫השתתף כסטודנט בפרויקט ח“ץ‬ ‫בין השנים ‪2010-2011‬‬ ‫‪[email protected]‬‬ ‫‪227‬‬

Cancer Letters 362 (2015) 149–157 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet Original Articles Reduced expression and growth inhibitory activity of the aging suppressor klotho in epithelial ovarian cancer Irina Lojkin a,b,1, Tami Rubinek a,1, Sandra Orsulic c, Omer Schwarzmann b, Beth Y. Karlan c, Shikha Bose d, Ido Wolf a,b,* a Institute of Oncology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel b Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel c Women’s Cancer Program, Cedars-Sinai Medical Center, Los Angeles, CA, USA d Department of Pathology & Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA ARTICLE INFO ABSTRACT Article history: Klotho is an anti-aging transmembrane protein, which can be shed and function as a hormone. Accu- Received 27 January 2015 mulating data indicate klotho as a tumor suppressor in a wide array of malignancies, and we identified Received in revised form 16 March 2015 klotho as an inhibitor of the insulin-like growth factor (IGF-1) pathway in cancer cells. As this pathway Accepted 17 March 2015 is significant in the development of epithelial ovarian cancer (EOC) we studied klotho expression and activity in this tumor. Klotho mRNA levels were reduced in 16 of 19 EOC cell lines and immunohisto- Keywords: chemistry analysis revealed high expression in normal ovaries, and reduced expression in 100 of 241 Ovarian cancer high grade papillary-serous adenocarcinoma of the ovaries, fallopian tubes and peritoneum. Reduced ex- Klotho pression was associated with wild-type BRCA status. Klotho reduced EOC cell viability, enhanced cisplatin Estrogen receptor sensitivity, and reduced expression of mesenchymal markers. Finally, klotho inhibited IGF-1 pathway ac- Tumor suppressor tivation and inhibited transcriptional activity of the estrogen receptor. In conclusion, klotho is silenced in a substantial subset of the tumors and restoring its expression slows growth of EOC cells and inhibits major signaling pathways. As klotho is a hormone, treatment with klotho may serve as a novel treat- ment for EOC. © 2015 Elsevier Ireland Ltd. All rights reserved. Introduction renal tubules of the kidney and the choroid plexus in the brain, and is also present in various exocrine and endocrine tissues, includ- Ovarian carcinoma is the second most common and the most ing the breast, pancreas and ovaries [8–11]. The extracellular domain deadly malignancy of the female reproductive tract [1]. Insulin- of klotho is composed of two internal repeats, KL1 and KL2, that like growth factor (IGF)-1 and its receptor (IGF-1R) play key roles share amino-acid sequence homology to β-glucosidase but lack glu- in regulating the normal biology of ovarian surface epithelial cells cosidase activity [9,12]. Klotho knockout mice exhibit a phenotype and have also been implicated in the transformation of ovarian car- resembling human premature aging, while overexpression of klotho cinoma cells. Thus, IGF-1 serum levels may correlate with epithelial in mice extends life span [9,12]. Klotho is an important regulator ovarian cancer (EOC) risk; IGF ligands and IGF-1R are overexpressed of the fibroblast growth factor (FGF) pathway and is a mandatory in EOC tissues and their expression may be associated with adverse co-factor for the interaction of FGF23 with FGFRs 1,3 or 4 [13,14]. prognosis [2,3]. Furthermore EOC cells display an autocrine growth Another important activity of klotho is inhibition of the IGF-1R loop mediated through the IGF-1R [2,4]. Another factor that plays [8,10,12]. a role in ovarian tumorigenesis is estradiol and its receptor, estro- gen receptor (ER) [5,6]. ER is expressed in the majority of EOCs and We have previously identified klotho as a tumor suppressor in expression of estrogen-regulated genes may predict shorter overall breast and pancreatic cancers. We noted high klotho expression in survival in patients with high-grade serous carcinoma [7]. normal breast and pancreatic tissues whereas klotho was down- regulated, partly due to promoter hypermethylation, in the majority Human klotho is a 1012-amino acid single-pass transmem- of cancerous tissues [8,10,15,16]. Overexpression of klotho or treat- brane protein, which is abundantly expressed in the distal convoluted ment with the soluble protein inhibited the IGF-1 pathway and slowed the growth of breast and pancreatic cancer cells in vitro and * Corresponding author. Tel.: +972 52 736 0558; fax: +972 3 697 3030. in vivo [8,10]. Recently, numerous studied established the role of E-mail address: [email protected] (I. Wolf). klotho as a potent tumor suppressor in a wide array of malignan- cies, including melanoma, colon, gastric, cervix, lung and renal cell 1 These authors contributed equally to the manuscript. cancers [17–26]. http://dx.doi.org/10.1016/j.canlet.2015.03.035 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved. 228

150 I. Lojkin et al./Cancer Letters 362 (2015) 149–157 The role of klotho in EOC has not been determined yet. Re- weeks cells were washed twice with PBS and colonies were stained with crystal violet cently, we discovered that a less active functional variant of KLOTHO and photographed. may be associated with increased risk of breast and EOC among BRCA1 mutation carriers [16]. A single study examined klotho mRNA 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay levels in EOC and noted reduced levels in 30% of tumors, but ex- pression was not tested in human normal ovaries [27]. While high For the assay, 3000 cells/well were plated in 96-well plates, cultured in the ap- expression was noted in mice ovaries [9], to our knowledge, klotho propriate culture media, and treated for 48 hrs as indicated. MTT assay was carried expression has not been studied in normal human ovaries. out as done previously [8]. We aimed to investigate klotho expression pattern in human Luciferase assay normal ovary tissue compared to the expression in high grade papillary-serous cancers of the ovary, peritoneum and fallopian tube. Cells were plated in 12-well plates and transfected with the reporter vector and Our results reveal high expression of klotho in normal ovarian ep- the klotho expression vector or pcDNA3 empty vector. Luciferase assay was con- ithelium and reduced expression in 41% of EOC samples. Expression ducted using Luciferase Assay System kit (Promega, CA) according to the of klotho, or treatment with soluble klotho, inhibited proliferation manufacturer’s instructions. Luciferase units were normalized to protein concentration. of EOC cell lines, enhanced their sensitivity to cisplatin and sup- pressed expression of mesenchymal markers. Finally, treatment with Statistical analysis klotho inhibited IGF-1 and ER signaling. Taken together, these data indicate klotho as a tumor suppressor in a subgroup of high grade Results are presented as mean ± standard deviation (SD). Categorized variables papillary-serous cancers. were compared between the study groups using Fisher’s exact test and continuous variables were compared using t-test. All significance tests were two-tailed and a P-value of <0.05 was considered as statistically significant. Materials and methods Results Chemicals, antibodies and constructs Klotho expression in ovarian cancer The chemicals used were IGF-1 (PeproTech Inc, Rocky Hill, NJ), cisplatin (Sigma, We first used quantitative RT-PCR and analyzed klotho mRNA St. Louis, MO), and estradiol (Sigma). The term soluble klotho, used throughout the levels in 19 human EOC cell lines, compared to the immortalized text, refers to soluble human klotho, encoding the amino acids 34–981 (R&D Systems, mammary cell line HB2 (high expressors) and the two breast cancer McKinley Place NE, MN). Antibodies: anti-diphosphorylated and -total ERK1/2 and cell lines MCF-7 (low expressors) and MDA-MB-231 (no expres- anti-β-actin (Sigma), anti-klotho for immunohistochemistry (IHC) (Calbiochem, BD sion) [15]. Klotho levels are presented in Fig 1a. No detectable or Biosciences, San Jose, CA) and for western blot anti-klotho (KM2076) was a kind gift very low levels of klotho (similar to MDA-MB-231 cells) were noted from Kyowa Hakko Kogyo Co., Ltd. Anti E-cadherin (Abcam, Cambridge, MA), anti- in 11 cell lines (TOV 112D, OV90, OV2008, CSOC 1031, ES2, PA1, ERα (F-10) and anti-progesterone receptor (PR) (C-20) (Santa Cruz Biotechnology, A2780, OVCA-432, OVCAR3, OVCAR5 and OVCAR8), low expres- Santa Cruz, CA). Expression vector containing the full length klotho (expressing the sion (similar to MCF-7 cells) was noted in five cell lines (TOV12G, entire coding region) was a generous gift of Y. Nabeshima (Kyoto University, Japan). 3A, UWBI-289BRCA1, CaOV4 and SKOV-3), whereas high expression Truncated klotho construct KL1 was described [8]. The ERE-luciferase reporter con- (similar to HB2 or higher) was noted in three cell lines (CSOC882, struct, kindly provided by D. Harris, (UCLA, CA), consists of 2 repeats of the upstream CaOV3 and UWBI-289VECTOR). We also tested klotho protein expres- region of the vitellogenin ERE promoter. sion in SKOV-3, ES2 and OVCA-432 cell lines and observed a similar protein and mRNA expression pattern (Fig 1b). No correlation was Tissue arrays and immunohistochemistry analysis noted between differentiation status, mutation status (including p53, KRAS, NRAS, PIK3CA) or other known characteristics of these cells Ovarian cancer tissue arrays were created, after IRB approval, from cancers di- and klotho expression. agnosed at Cedars-Sinai Medical Center from 1996 to 2007. Clinical parameters and follow-up information were obtained from hospital records. The tissue arrays were We next conducted IHC analysis and examined klotho expres- constructed using a manual arrayer (Beecher Instruments, MD) and contained three sion in a tissue array consisting of 24 normal ovary samples 1 mm samples for each specimen. Immunohistochemistry analysis was conducted compared to 176 EOC samples, 55 primary peritoneal cancers and as described earlier [8,10,15]. Staining was scored by percent of positive tumor cells 10 fallopian tube cancers (Table 1). All tumors were of papillary- and staining intensity. serous adenocarcinoma histology and most were of stage III and grade 3. High klotho expression was observed in all 24 normal ovary Cells and transfections samples while reduced expression was noted in 41% of the tumors (Fig 1c, Table 1, p < 0.005 for the comparison between normal and The EOC cell lines CSOC882, CaOV3, TOV 21G, OV 2008, 3A, UWB1-289VECTOR, each of the cancer groups). As 89% of all tumors were grade 3 and UWB1-289BRCA1, CaOV-4, A2780, SKOV-3, OVCA432, OVCAR-3, OVCAR-5, TOV 112D, 81% were stage III, the association between klotho and stage or grade OV-90, CSOC 1031, ES2, PA1, OVCAR-8 and the breast cancer cell lines MCF-7 and could not be determined using this cohort. MDA-MB-231 were obtained from the American Type Culture Collection (Manassas, VA). HB2, a non-tumorigenic mammary epithelial cell line, was developed by Dr. J. Germ-line BRCA1 (185delAG, 5382insC) and BRCA2 (6174delT) Taylor-Papadimitriou [28]. All transfections used JetPei (Polypus Transfection, Illkirch, mutation status was available for 83 of the cancer patients (Table 2). France). Similar tumor types were noted among BRCA1/2 mutation carri- ers and non-carriers. Higher klotho expression was noted among Quantitative reverse transcription-PCR (RT-PCR) BRCA1/2 mutation carriers compared to non-carriers (Table 2, p = 0.02). While these differences were similar for BRCA1 and BRCA2 Total RNA extraction, cDNA production and quantitative RT-PCR were per- carriers, significance was reached only for BRCA1 mutation carri- formed as described [29]. Primers used are presented in Supplementary Table S1. ers (p = 0.04) but not for BRCA2 mutation carriers (p = 0.14), probably due to the small number of patients in the latter group. Western blot analysis Klotho inhibits growth of ovarian cancer cells Protein extraction and Western blotting was performed as described [29]. The effect of klotho and its active domain KL1 on the growth of Colony assays EOC cells was first assessed using colony formation assays. OVCA-432 Two days following transfection with the indicated plasmids, G418 (750 μg/ml for SKOV-3 and for OVCA432 and 900 μg/ml for ES2 cells) was added to the culture media. At day 14, cells were stained using crystal violet (Sigma) and quantitated as described [8,10]. For colony assays with cisplatin, 300 cells were seeded in 6 well plates, and after 24 hrs cells were treated with klotho, cisplatin, their combination or control vehicle. Media and treatments were changed twice a week, and after two 229

I. Lojkin et al./Cancer Letters 362 (2015) 149–157 151 Fig. 1. Klotho expression is reduced in ovarian cancer. (a) Klotho mRNA levels were determined by quantitative RT-PCR in EOC cell lines and were compared to the expres- sion in breast cancer cell lines (MCF-7 and MDA-MB-231) and to the non-tumorous mammary cell line HB2. (b) Klotho protein expression in EOC cells was determined using Western blot. (c) Reduced klotho expression in ovarian cancer tissue array. Immunohistochemistry staining of klotho in representative tissue samples of normal ovary tissue, showing high klotho expression, and of ovarian cancer with high klotho expression and a cancer tissue with low klotho expression (all at ×100 and ×400 magnifications). 230

152 I. Lojkin et al./Cancer Letters 362 (2015) 149–157 Table 1 Surprisingly, in OVCA432 cells, which were resistant to cisplatin, Klotho expression and clinical and pathological characteristics of the study group. treatment with cisplatin abrogated klotho growth inhibitory effect (Fig 2c). Normal Ovarian Fallopian Peritoneal ovaries cancer tube cancer cancer Klotho suppresses mesenchymal marker in ovarian cancer cells n = 24 n = 176 n = 10 n = 55 n (%) n (%) n (%) n (%) Klotho has been shown to inhibit EMT in lung and cervix cancer cells. We, therefore, studied this activity in EOC cells. Klotho Age 37–73 34–89 40–87 38–86 overexpression increased E-cadherin protein expression in OVCA432, Range 55 61 61 67 SKOV-3 and ES2 cells (Fig 3a). This increase was also observed in Median the mRNA level in OVCA432 and SKOV-3 cells (Fig 3b-d, p < 0.001 24 (100) 12 (7) 0 (0) 2 (3) and p < 0.005, respectively). Klotho also decreased mRNA expres- Grade 0 (0) 164 (93) 10 (100) 51 (94) sion of the mesenchymal marker Snail1 in the tested cell lines G2 (Fig 3b-d) and of Snail 2 in two of them. Vimentin was G3 0 (0) 0 (0) 2 (3) downregulated by klotho only in OVCA432 cells (Fig 3b, p < 0.001). Unknown 5 (3) 1 (10) 0 (0) Klotho suppresses activation of the IGF-1 pathway in ovarian cancer Stage 7 (4) 1 (10) 0 (0) cells I 136 (77) 8 (80) 51 (93) II 27 (15) 0 (0) 4 (7) The IGF-1 pathway plays an important role in the develop- III 1 (1) 0 (0) 0 (0) ment of EOC [31] and klotho inhibits activation of this pathway in IV other malignancies [8,10]. We examined the ability of klotho to Unknown 108 (61) 6 (60) 27 (41) inhibit activation of the IGF-1 pathway in OVCA432 and SKOV-3 cells. 68 (39) 4 (40) 28 (51) For these studies, cells were transfected with either klotho expres- Klotho expression* sion plasmid or an empty vector, treated with IGF-1 (15 min), and Normal pathway activation as assessed by ERK1/2 phosphorylation was ana- Reduced lyzed using Western blotting. Klotho over-expression reduced IGF- 1-induced ERK1/2 phosphorylation in both cell lines (Fig. 4a). * p < 0.005 for the comparison between normal ovaries and each of the other groups. Klotho inhibits the ERα pathway in ovarian cancer cells and ES2 cells (no klotho expression, Fig 1a,b) and SKOV-3 (moder- ate klotho expression) were employed for these assays. Cells were The ER plays an important role in the development of EOC and transfected with either full length klotho plasmid, the KL1 domain is a downstream effector of IGF-1 signaling in EOC [7]. Studies in or an empty vector, cultured in media containing G418 for two weeks breast cancer cells indicate klotho as a modulator of ER activity (un- and stained to determine the number of surviving colonies. Klotho published data). We, therefore, evaluated klotho effect on the ER or KL1 expression reduced the number and size of surviving colo- pathway in EOC cells. We first examined the effect of klotho on the nies in all cell lines tested (Fig. 2a). transcriptional activity of the ER using ERE-luciferase reporter. For these studies, SKOV-3 and OVCA432 cells were co-transfected with Klotho may be shed and act as a circulating hormone, and we the ERE-luciferase reporter and either klotho expression plasmid and others have shown that soluble klotho can affect cellular ac- or an empty vector, and treated with either a vehicle control or E2. tivities [8,10]. We, therefore, tested the ability of soluble klotho to Treatment with E2 increased ERE activity by 2.5-fold in SKOV-3 cells inhibit proliferation of EOC cells. Cells were treated with elevated and by 20% in OVCA432 cells, while treatment with klotho inhib- klotho concentrations for 48 hrs, and viability was assessed by MTT ited E2-mediated transcriptional activity (Fig 4b, p < 0.01). Further assay. Treatment with klotho (100 ng/ml) reduced viability of OVCA- studies in SKOV-3 cells revealed that klotho inhibited E2-mediated 432, ES2 and SKOV-3 cells by 40%, 22% and 20%, respectively (Fig 2b, mRNA upregulation of the ER regulated genes, TGFα and the PR p < 0.005 for klotho 0.1 ng/ml for all cell lines). (Fig 4c) and also reduced protein levels of the PR (Fig 4d). We have shown that klotho enhances sensitivity of pancreatic Discussion cancer cells to gemcitabine, and others have shown that it sensi- tizes lung cancer cells to cisplatin [8,30]. Platinum-based This study indicates klotho as a tumor suppressor in EOC. Klotho chemotherapeutic drugs are the mainstay of treatment for EOC [1]. is highly expressed in the normal ovary but its expression is reduced In order to test whether klotho enhances sensitivity of EOC cells to in 40% of the Mullerian cancers. Furthermore, klotho slowed pro- cisplatin, cells were treated with soluble klotho, cisplatin, their com- liferation of EOC cells and inhibited major growth-promoting bination, or a control vehicle and cell viability was measured. At a signaling pathways. concentration of 3.2 μM, cisplatin reduced viability of SKOV-3 cells by 27% and of ES2 cells by 35%, while the addition of klotho further Klotho is expressed in a wide array of normal endocrine and exo- reduced viability of these cells by 40% and 52%, respectively (Fig 2c, crine tissues [8,9,11,15]. Our data indicate high klotho expression p < 0.05). Moreover, studies in SKOV-3 cells revealed that com- in the normal human ovary. The role of klotho in the normal ovary bined treatment with klotho and cisplatin significantly reduced has not been determined yet. Klotho deficient female mice are sterile colony number compared to either treatment alone (Fig 2d). and their follicles do not mature beyond the prenatal stage [32]. Yet, the mechanisms governing the activities of klotho in the ovaries Table 2 remain to be elucidated. Accumulating data indicate silencing of it Klotho expression by BRCA mutation status. in breast, pancreas, cervix, lung, colon and gastric cancers, as well as in melanoma [8,15,17,22,24,25,33]. We noted reduced klotho levels Wild type All BRCA BRCA1 BRCA2 in 40% of all tumors. A previous study analyzed klotho mRNA ex- BRCA mutations mutations mutations pression in 189 EOC samples and noted silencing of klotho in 30% n = 42 N = 41 n = 25 n = 16 of samples [27]. Of note, the study did not include normal samples n (%) N (%) n (%) n (%) Tumor type 27 (64) 33 (80) 18 (72) 15 (94) Ovarian 3 (7) 4 (10) 3 (12) 1 (6) Fallopian 4 (10) 4 (16) 0 (0) Peritoneal 12 (29) 29 (71) 18 (72) 11 (69) Klotho expression* 19 (45) 12 (29) 7 (28) 5 (31) Normal 23 (55) Reduced * p = 0.02 for the comparison between wild type and all mutated BRCA, p = 0.04 for wild type vs. BRCA1 mutated and p = 0.14 for wild type vs. BRCA2 mutated. 231

I. Lojkin et al./Cancer Letters 362 (2015) 149–157 153 Fig. 2. Klotho inhibits growth of ovarian cancer cells and sensitizes them to cisplatin. (a) EOCs were transfected with either klotho or KL1 expression vectors or an empty vector (pcDNA3). Transfected cells were cultured in media containing G418 for two weeks, and then colonies were stained with crystal violet and photographed. (b) EOCs were plated in 96-well plates and treated with either human klotho at elevated concentrations or a vehicle control. Viability was assessed after 48 hrs using MTT assay. Data are shown as mean ± s.d. of three independent experiments. * p < 0.05 and ** P < 0.005 for comparison between treated cells vs. control. (c) EOCs were plated in 96- well plates and treated with either soluble klotho, cisplatin, their combination or a control vehicle. MTT assay was conducted at 48 hrs. Data are shown as mean ± s.d. of three independent experiments. * p < 0.05 and ** p < 0.005. (d) SKOV-3 cells were seeded at low density (300 cells per 35 mm well) and treated with soluble klotho (1 ng/ml), cisplatin (0.5 μM), and their combination or control vehicle, for 2 weeks. Colonies were stained with crystal violet and photographed. and did not analyze klotho protein expression. While klotho silenc- Our studies indicate klotho as a potent inhibitor of proliferation ing is a near universal event in some tumors (i.e. breast and of EOC cells. Interestingly, klotho showed a similar effect on SKOV-3 pancreatic caners), it was noted in only a subgroup of other tumors cells, which express high klotho levels, and on OVCA432 and ES2 (i.e. cervical cancer, colon cancer) [22,24]. We noted lower klotho cells which express very low klotho levels. Similarly, the pancreatic expression among non-carriers of BRCA1/2 mutation. This obser- cancer cell line Panc1 expressed high klotho levels, yet exhibited vation suggests that klotho silencing may be specifically required similar in vitro and in vivo sensitivity to that of the low expressor for a subgroup of EOCs that is not driven by altered BRCA1/2 activity. MiaPaca2 [8]. Klotho activity is dependent, among other mecha- nisms, upon its shedding, which is regulated by Disintegrin and In some tumors, including pancreatic, lung, renal, gastric, and metalloproteinase domain-containing protein (ADAM) 10 and 17 [38]. hepatocellular carcinoma, reduced klotho expression or klotho pro- While high ADAM 10 expression was noted in A2780 EOC cells and moter hypermethylation was associated with poor prognosis also in primary EOC [39], to our knowledge, its expression in other [21,34–37]. As in our study most tumors were of grade 3 and stage EOC cells has not been studied. It remains to be determined whether III, analysis of the association between klotho and adverse tumor reduced expression of ADAM 10 or 17 is responsible for the relatively characteristics could not be performed. 232

154 I. Lojkin et al./Cancer Letters 362 (2015) 149–157 Fig. 3. Klotho regulates epithelial and mesenchymal markers in ovarian cancer cells. (a) EOCs were seeded and transfected with klotho or control empty vector, and 48 hrs after transfection cells were harvested and E-cadherin protein expression was evaluated or RNA was extracted and E-cadherin, vimentin, snail1 and snail2 were deter- mined in (b) OVCA432, (c) SKOV-3 or (d) ES2 cells, using quantitative RT-PCR. *, p < 0.05; **, p < 0.005; ***p < 0.001, for klotho transfected compared to pcDNA transfected cells. reduced activity of klotho in cells that express high klotho levels. implicated in the transformed phenotype of ovarian carcinoma cells. Regardless of the mechanism, our data indicate that exposure of Indeed, inhibition of the IGF-1R, either using small molecules or hu- cancer cells to supra-physiologic concentrations of klotho can inhibit manized antibody, inhibits proliferation EOC cells, and several phase proliferation of EOC irrespective of the levels of endogenous klotho. II and III clinical trials using different strategies for IGF-1R inhibi- tion are currently being conducted (summarized in Ref. 31). Klotho Our data indicate that klotho not only affects proliferation but is a potent inhibitor of the IGF-1 pathway and we show here that also inhibits EMT markers in EOC cells, as indicated by inhibition klotho inhibits IGF-1-mediated activation of ERK1/2. A similar ac- of Snail 1 and 2 expression and up-regulation of E-cadherin. Inhi- tivity of klotho has been shown by us, as well as by other groups, bition of EMT or its markers by klotho has also been demonstrated in breast, pancreatic, lung, gastric and hepatocellular cancer cells in lung and cervical cancer cells [18,20]. [8,10,19,21,40]. The mechanisms mediating this activity of klotho have not been elucidated. Klotho can bind the IGF-1R but it does The IGF-1 and its receptor play key roles in regulating the normal biology of ovarian surface epithelial cells and have been 233

I. Lojkin et al./Cancer Letters 362 (2015) 149–157 155 Fig. 4. Klotho inhibits the IGF-1 and estrogen receptor pathways in ovarian cancer cells. (a) Cells were transfected with either a klotho expression plasmid or a control vector (pcDNA3); after 24 hrs, cells were serum starved for 48 hrs and treated for 15 min with IGF-1 (50 ng/ml). Following treatment, cells were harvested and proteins were resolved and immunoblotted using anti phospho (p) or total (t) ERK1/2 antibodies. Klotho inhibits ER transcriptional activation in ovarian cancer cells. (b) Cells were grown in phenol-free medium and charcoal-stripped serum, then transfected with klotho or control vector, together with the ERE-luciferase reporter. Twenty-four hrs later cells were treated with E2 (10 nM) as indicated, for 24 hr. The luciferase activities were analyzed and normalized to total protein concentration, and are shown relative to the control vector. (c) SKOV-3 cells were transfected and treated as in (b), and PR and TGFα mRNA levels were determined 48 hrs after transfection by qRT-PCR. The figure shows representative results of three independent experiments, each performed in triplicates. *, p < 0.05; **, p < 0.005. (d) SKOV-3 cells were transfected as in (b), but grown in regular medium (FBS and phenol-red) and 48 hrs later PR expression was analyzed using Western blot. The figure shows a representative figure of three independent experiments. not prevent ligand binding, and unlike other activities of klotho, IGF- of IGF-1. Indeed, IGF-1 mRNA expression was noted in primary and 1R inhibition can be mediated by the KL1 domain alone [8,12]. metastatic ovarian cancer samples [3]. Interestingly, klotho mRNA expression correlated with elevated ex- pression of IGF-1 in EOC clinical samples [27]. It is, therefore, possible Platinum-based chemotherapies play a central role in the therapy that cells which express klotho evade its growth inhibitory effects of EOC [1]. Emergence of platinum resistance is a major obstacle by upregulating survival mechanism, including enhanced production in the treatment of EOC patients and the IGF-1R may participate in this process [41]. We show that klotho sensitizes SKOV-3 and ES2 234

156 I. Lojkin et al./Cancer Letters 362 (2015) 149–157 cells to cisplatin. Similar results were also observed in the human [4] S. Hirano, N. Ito, S. Takahashi, T. Tamaya, Clinical implications of insulin-like lung cancer cell line A549, where klotho sensitized cells to cisplatin growth factors through the presence of their binding proteins and receptors via inhibition of the PI3K/Akt pathway [30]. In lung cancer cells, expressed in gynecological cancers, Eur. J. Gynaecol. Oncol. 25 (2004) 187–191. klotho expression inversely correlated with resistance to cisplatin. This is not observed in EOC cells, where both cell lines with low [5] F. Mungenast, T. Thalhammer, Estrogen biosynthesis and action in ovarian klotho (ES2 and OVCA432) exhibit high and low sensitivity to cancer, Frontiers Endocrinol. 5 (2014) 192. cisplatin. Thus, more factors play a role in conferring sensitivity to cisplatin. [6] G.G. Rao, D.S. Miller, Hormonal therapy in epithelial ovarian cancer, Expert Rev. 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