DNA Demethylation Regulates Gene Expression in Human Intervertebral Disc Cells

Proceedings of the NASS 27th Annual Meeting / The Spine Journal 12 (2012) 99S–165S PATIENT SAMPLE: Patients 18-65 years of age with degenerative disc disease at one level between C3 and T1with neurologic symptoms unresponsive to conservative care were included. Prior adjacent-level fusions were allowed. The per-protocol patient sample at 2 years included 395 patients (211 PCM, 184 ACDF control). 340 patients had available data for subgroup analyses, including 45 WC (26 PCM, 19 ACDF) and 295 NC (163 PCM, 132 ACDF). OUTCOME MEASURES: Clinical outcome measures, including the validated patient-reported neck disability index (NDI), maintenance or improvement of neurological status, and complications and adverse events were evaluated. METHODS: Overall success was defined as a $20% improvement in NDI, maintenance or improvement in neurologic status, no subsequent secondary surgery or intervention (SSSI), absence of major adverse events, and absence of radiologic complications. Where appropriate, measures were tested for differences between arthroplasty and fusion groups and between WC and NC groups at 2 years postoperative. RESULTS: Demographic and baseline characteristics were well matched between WC and NC patients except on presence of litigation related to spinal disorder, which was notably higher in WC patients. At 2 years, regardless of treatment group, WC patients compared to NC patients had a significantly lower rate of NDI success (67% vs. 84%, p50.011), absence of SSSI (87% vs. 95%, p50.044), and overall success (53% vs. 73%, p50.008), respectively. Success of WC patients compared to NC patients on absence of major complication (98% vs. 99.7%), neurological success (86% vs. 93%), and radiographic success (98% vs. 95%), were similar. Comparing PCM patient outcomes by compensation status, overall success was met in 58% of WC and 78% of NC patients (p50.048) and NDI success was met in 65% and 86% (p50.021), respectively. Other outcomes were similar: SSSI success was met in 88% of WC and 95% of NC patients, neurological success was met in 92% and 95%, radiographic success was met in 100% and 99%, and major complications success was met in 100% and 100%, respectively. CONCLUSIONS: Confirming the results of other studies, the pursuit of workers compensation claims negatively affects patient success, as demonstrated in both the current PCM and control groups. Lack of success was primarily seen in patient-reported subjective measures (PCM NDI success: 65% WC vs. 86% NC), where objective measures (SSSI, neurologic, radiographic, and major complications) were largely equivalent. FDA DEVICE/DRUG STATUS: PCM (Investigational/Not approved).

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STUDY DESIGN/SETTING: This is a morphologically based, prospectively designed, single-center study. PATIENT SAMPLE: Thirty consecutive patients (14 male, 16 female) who treated for degenerative spinal pathologies were included in the study. Patients with cervical fractures, occluded VA, prominent degenerative changes affecting TF, deformity, or previous cervical instrumentation were excluded from the study. OUTCOME MEASURES: In all patients, computed tomography (CT) of the cervical spine and doppler ultrasonography of the VAs were obtained for morphometric analysis. METHODS: Axial CT cuts were taken using Toshiba Aquilion 64 (Toshiba Medical Systems). Two measurements were performed for each foramen: its width (its maximum right-left diameter) and its depth (its maximum antero-posterior diameter). Measurements of bilateral C6 transvers foramina were subjected to statistical analysis for consistency. Blood flow volume of bilateral VAs were measured using 3D color doppler with either a Logiq 7 or Logiq 9 system (GE Healthcare, Milwaukee, WI). Relationship between sizes of the transvers foramen and blood flow of corresponding VA were subjected to statistical analysis. RESULTS: Diameters of the TF was around 5 mm (ranging 2,2 to 7 mm), being its width was slightly larger than depth. Transvers foramina were always asymmetric, with no right or left side preference. There was a strong correlation between TF diameters and blood flow of the VAs. Between TF width and VA blood flow Pearson’s correlation coefficient was 0,59 (P50,001) for right side, and 0,72 for left side (P!0,0001). Correlation between TF depth and blood volume was 0,52 for right side (P50,004) and 0,58 (P50,001) for left side. The side of the larger TF matched with the side of dominant VA in 28 out of 30 cases (93%) (P!0,0001). The odds ratio for this agreement is 195 for TF width, and 182 for TF depth. CONCLUSIONS: We found that there was strong correlation between TF diameters and VA blood volume. As the main goal of the study, we also found that in 93% of the patients, side of the larger TF and dominant VA were the same. Thus, the current study suggests that the size of the transvers foramina can be used reliably to estimate the side of the dominant VA (odds ratio is 195). This relationship may obviate the necessity to perform any additional imaging technique to determine side of the dominant VA in most cases. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. http://dx.doi.org/10.1016/j.spinee.2012.08.417

http://dx.doi.org/10.1016/j.spinee.2012.08.416

P143. Sizes of the Transvers Foramina Correlate with Blood Flow and Dominancy of Vertebral Arteries Kadir Kotil, MD1, Cumhur Kilincer, MD, PhD2; 1Haseki Training & Research Hospital, Istanbul, Turkey; 2Trakya University Medical Department of Neurosurgery, Edirne, Turkey BACKGROUND CONTEXT: The diameters of the vertebral arteries (VAs) are very often unequal, and the left VA is often larger than the right VA. Recent studies have suggested that unequality of the VAs may have certain hemodynamic effects on the vertebro-basilary system. Moreover, knowing the side of the dominant VA may be of utmost importance if the VAs are at risk during spine surgery.Determination of the size of the VAs is generally obtained by the utilization of angiography (digital substraction, or computed tomography, or magnetic resonance angiographies) or by doppler ultrasonography. PURPOSE: Because the VA is the main anatomic structure occupying the TF, it may be expected that size of the TF and blood flow of VAs should be proportional. The current paper investigates a novel idea of there is a correlation between the size of the TF and the blood flow and dominancy of the VA. Existence of such a correlation may help the surgeon to predict the side of the dominant VA without performing any additional imaging technique.

P144. DNA Demethylation Regulates Gene Expression in Human Intervertebral Disc Cells Sadanand Fulzele, PhD, DVM, Norman Chutkan, MD; Medical College of Georgia, Augusta, GA, US BACKGROUND CONTEXT: The intervertebral disc (IVD) cell distribution depends on the developmental stage of the human disc. DNA methylation has been shown to be essential to mammalian development, and aberrant methylation patterns are a common feature of many kinds of age-related diseases. We hypothesize that the methylation status of the promoter region of the genomic DNA regulates gene expression of IVD cells. PURPOSE: To analysis the change in gene expression following experimentally-induced loss of DNA methylation of Human IVD cells. METHODS: IVD tissues were obtained from donors undergoing anterior lumbar discectomy procedures with Institutional Review Board approval. The tissues were transported to the laboratory, minced, and immediately underwent cell isolation by sequential enzyme digestion. The digested cells were centrifuged and seeded in culture dishes, supplemented with complete medium (DMEM/F12) containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% antibiotic. The demethylation agent 5azacytidine (5-aza-C) was used to inhibit DNA methylation in IVD cells. The cells were then plated and treated with 5-aza-C followed by RNA isolation and real time PCR for gene analysis.

All referenced figures and tables will be available at the Annual Meeting and will be included with the post-meeting online content.

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Proceedings of the NASS 27th Annual Meeting / The Spine Journal 12 (2012) 99S–165S

RESULTS: As a pilot study, we investigated cytokines (IL-1b, IL-6), anabolic genes (Aggrecan, Collagen type II apoptotic (Bax), and cell cycle (p16, cyclin D2) genes. In the presence of 5-azaC treatment, IL-1b, IL6, Bax, p16, and cyclin D2 expression were upregulated ~2 fold or more. The anabolic gene aggrecan was down regulated whereas no effect on Collagen type II expression. CONCLUSIONS: The experimentally-induced loss of DNA methylation results in the increase expression of apoptoticgene expression.Furthermore, demethylation results in both increased gene transcription of catabolic and inflammatory genes as well as a down regulation of aggrecan, thus confirming a cause-and-effect relationship between DNA demethylation and transcription of these genes in IVD cells. DNA demethylation has been previously shown to inhibit cell proliferation (5) and induce apoptotic cell death in different cell types. Our results suggest that demethylation of IVD cells may be one of the influential causes for IVD degeneration. Further studiesare requiredto confirm our findingsand to better our understanding of the effects of aberrantDNA methylation on IVD biology and disease pathophysiology. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. http://dx.doi.org/10.1016/j.spinee.2012.08.418

P145. 3D Ultrasound Imaging for Assessment of Scoliosis Yong-Ping Zheng, PhD; The Hong Kong Polytechnic University, Hong Kong, China BACKGROUND CONTEXT: Adolescent idiopathic scoliosis (AIS)is defined as deformity of the spine in coronal plane with more than 10 degrees of Cobb’s angle, which measures from the end-plates of the end vertebral bodies in a standing radiograph. It is accompanied by deformities in the sagittal plane together with axial rotational deformities. About 3.1% children suffer from scoliosis in Hong Kong, and similar number has been reported in other regions. The outcome measure for the treatment of patients with AIS relies heavily on the monitoring of coronal and sagittal curves, progression of deformity, and conservative and surgical management. Cobb’s angle is the gold standard in monitoring patients susceptible to scoliosis development. Patients need to conduct an X-ray examination of theirwhole spine regularly every 4 to 6 months until skeletal maturity is reached. However, it was reported that there are only less than 1 % of the screened population and less than 10% of those with curves greater than 10 degrees warrant treatment. This previous finding implies that more than 90% of patients are subject to unnecessary radiation. It is clearly necessary to have a system that can provide AIS mass screening and longitudinal follow-up for children without the hazard of radiation. PURPOSE: The objective of this study is to develop a radiation-free method for the measurement of spine deformity based on 3D ultrasound imaging and to demonstrate its feasibility with the tests on subjects with scoliosis. STUDY DESIGN/SETTING: This is a study related to the development of a new measurement system for scoliosis assessment using 3D ultrasound imaging. PATIENT SAMPLE: Twenty-seven subjects with various degrees of scoliosis were recruited with the help of a Chinese Medicine practitioner, who provides massage treatments to these subjects. OUTCOME MEASURES: The correlation between the Cobb’s angles measured using conventional X-ray method and 3D ultrasound method was conducted. The intra-operator reproducibility of the measurement was also carried out. METHODS: A 3D ultrasoundimaging system was developed for the assessment of scoliosis. The system consists of a portable ultrasound scanner, a frame structure, an electromagnetic spatial sensing device, and a PC with program for image collection, analysis, and display. Before scanning, four body supporters, which were mounted on the cheat board with their positions carefully adjusted to ensure body immovable. B-mode ultrasound images (2D) and their spatial information were recorded simultaneously

during scanning. These images collected from subjects were displayed in 3D space corresponding to the spatial information collected. The frames which consist of spinous or traverse processes were selected for marking the processes’ position. Since the B-mode image maynot clearly display the bony surface, image processing methods were applied for enhancing the bony surface. The tip of each process was then manually assigned with a marker. Based on these markers, a 3D model of spine was formed and used to measure the spine deformity angle, i.e. Cobb’s angle. The system further provided a function to fuse ultrasound images with X-ray images for Cobb’s angle measurement. To test the reproducibility, both X-ray and 3D ultrasound images were reviewed by the same operator twice. RESULTS: According to the results obtained from the 27 subjects, the correlation between the Cobb’s angles measured using the X-ray method and 3D ultrasound method was very high (R250.8621). The measured Cobb’s angle for these subjects ranged from 0.8 to 25.9 degrees. The intra-operator reproducibility of the measurement methods were high with R2 of 0.9321 and 0.8425 for X-ray and 3D ultrasound measurements, respectively. CONCLUSIONS: It was demonstrated that the 3D ultrasound imaging method can provide a reliable measurement for the Cobb’s angle for scoliosis without radiation hazard, with accuracy and reproducibility similar to conventional X-ray method. It is suitable for close monitoring of the progression and treatment outcomes for scoliosis as well as for mass screening for adolescent idiopathic scoliosis. Further clinical evaluation with a larger patient sample number is required for evaluating this new technique. FDA DEVICE/DRUG STATUS: 3D ultrasound imaging system for scoliosis assessment (Not approved for this indication). http://dx.doi.org/10.1016/j.spinee.2012.08.419

P146. Distinguishing Benign Notochordal Cell Tumor from Chordoma Jennifer Kreshak, MD, Frederique Larousserie, MD, Stefano Boriani, MD, Piero Picci, MD, Joseph Mirra, MD, Daniel Vanel, MD; Istituto Ortopedico Rizzoli, Bologna, Italy BACKGROUND CONTEXT: Benign notochordal remnants have been reported in the nucleus pulposus and as small extraosseous soft tissue masses, ecchordosis physalifora. Similar entities have been found in vertebrae, but only recently well characterized. They appear to be more prevalent than originally recognized and are thought to possibly be precursors to chordoma. Terminology has varied; they are now referred to as giant notochordal rests or benign notochordal cell tumors (BNCT). Histopathologically, they have morphological features distinct from the notochordal cells found in disks and from chordomas, however, differentiating between these can be difficult. They have consistently been described as radiologically distinct from chordoma, being undetectable or ‘sclerotic’ on radiographs and computed tomography and without evidence of bony destruction or soft tissue masses. PURPOSE: To better identify and characterize benign notochordal lesions and their possible relationship to chordomas. STUDY DESIGN/SETTING: Retrospective review. METHODS: All cases of chordoma diagnosed at our institution prior to 2008 (at which point BNCT was a well-recognized entity) were reviewed, yielding 174 cases. Of these, only five were without soft tissue masses; these five were reevaluated radiographically and histopathologically. RESULTS: One of the five cases was again diagnosed as a chordoma. Two were found to have areas of both chordoma and BNCT. Two were determined to be BNCT. All lesions had radiographic features consistent with chordoma, most notably areas of lysis on computed tomography and evidence of bony destruction. All patients had vertebrectomies; one chordoma recurred, one BNCT recurred as chordoma, all others have no evidence of recurrence. CONCLUSIONS: The true nature of BNCT continues to be controversial and is ever-evolving; there remains dispute as to whether it is a precursor to chordoma. Despite cases in the literature documenting no radiographic changes in BNCTs over years, chordomas have been found

All referenced figures and tables will be available at the Annual Meeting and will be included with the post-meeting online content.