Huddleston, P.M.

Mini We compared the sensitivity and specificity of peri-implant tissue culture to the vortexing–sonication technique for the diagnosis of spinal implant infection (SII). Lower thresholds of sonicate fluid culture positivity showed increased sensitivity with maintained specificity. We recommend a threshold of 20 CFU/10 mL for sonicate culture positivity for the diagnosis of SII. Study Design. This is a retrospective study comparing the diagnosis of spinal implant infection (SII) by peri-implant tissue culture to vortexing–sonication of retrieved spinal implants. Objective. We hypothesized that vortexing–sonication would be more sensitive than peri-implant tissue culture. Summary of Background Data. We previously showed implant vortexing–sonication followed by culture to be more sensitive than standard peri-implant tissue culture for diagnosing of SII. In this follow-up study, we analyzed the largest sample size available in the literature to compare these two culture methods and evaluated thresholds for positivity for sonicate fluid for SII diagnosis. Methods. We compared peri-implant tissue culture to the vortexing–sonication technique which samples bacterial biofilm on the surface of retrieved spinal implants. We evaluated different thresholds for sonicate fluid positivity and assessed the sensitivity and specificity of the two culture methods for the diagnosis of SII. Results. A total of 152 patients were studied. With more than 100 colony forming units (CFU)/10 mL as a threshold for sonicate fluid culture positivity, there were 46 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 65.2% and 79.6%; the specificities were 88.7% and 93.4%, respectively. With more than 50 CFU/10 mL as a threshold, there were 50 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 68.0% and 76.0%; the specificities were 92.2% for both methods. Finally, with more than or equal to 20 CFU/10 mL as a threshold, there were 52 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 69.2% and 82.7%; the specificities were 94.0% and 92.0%, respectively. Conclusion. Implant sonication followed by culture is a sensitive and specific method for the diagnosis of SII. Lower thresholds for defining sonicate fluid culture positivity allow for increased sensitivity with a minimal decrease in specificity, enhancing the clinical utility of implant sonication. Level of Evidence: 4

Abstract Abstract 1814 Multiple myeloma (MM) is an incurable hematologic disorder characterized by the accumulation of plasma cells (PCs) in the bone marrow (BM). Like normal B cells and PCs, MM cells express a distinctive immunoglobulin (Ig) molecular fingerprint resulting from heavy and light chain gene rearrangements and unique mutations introduced by the somatic hypermutation (SHM) process. Detailed examination of the Ig molecule may offer insight into the etiology of B cell malignancies as well as the ontogeny of malignant transformation and ongoing genetic evolution. Because prior studies of the Ig fingerprint of MM cells have been limited in size and scope, our current understanding of MM Ig repertoire is minimal. We used massively parallel sequencing of normal and malignant PCs to interrogate: 1) DNA versus RNA (cDNA) as starting material for studies of Ig repertoire; 2) the Ig repertoire of normal and malignant PCs; and 3) intraclonal heterogeneity in MM. Bone marrow PCs (BMPCs) from an untreated MM patient and from a normal control subject were isolated by magnetic bead separation. DNA and RNA were isolated from purified cells and amplified in a multiplex PCR reaction with primers designed to capture the entire IGHV region including the heavy chain complementarity determining region 3 (HCDR3) necessary for defining clonal cells. Using Roche 454 GS-FLX Titanium chemistry and a GS FLX Titanium Genome Sequencer, over 30,000 sequence reads were obtained from each sample. Following a detailed algorithm to minimize Taq and sequencing related errors and removal of non-Ig or non-productive Ig sequences, the resulting sequences were grouped based on HCDR3 amino acid identity. Each unique HCDR3 represented one member of the repertoire and all the sequences with the same HCDR3 defined the clone size. This measurement showed that there was nearly a five-fold higher level of unique HCDR3s in normal BMPCs when the starting PCR template was DNA vs. cDNA. Thus, DNA is clearly a better starting template for repertoire analysis using deep sequencing methods. Thorough analysis of the MM DNA sample identified PCs expressing 37 different IGHV genes, and predictably, the vast majority of sequence reads (83%) had exact nucleotide identity in the IGHV and HCDR3 region (IGHV3-74 with 18 mutations in the IGHV region) thereby defining the dominant MM clone. There were also ∼5000 additional sequences that either shared an identical HCDR3 region but varied within the IGHV3-74 gene sequence (n=3975) or were identical within the IGHV3-74 sequence yet varied within the HCDR3 (n=862). These sequences were further analyzed, with sequences not present 10 or more times or not found in both the 5' and 3' directions eliminated from further scrutiny. This process revealed a total of 64 putative subclones with single point mutations in the IGHV or HCDR3 region (10–56 copies of each unique sequence representing a total of 1583 clonally related sequences). When we used a more stringent subclone frequency of ≥0.1%, 22 subclones remained. Of note, conventional Ig repertoire analysis would require sequencing ≥1000 colonies to detect MM subclones present at this frequency. Finally, 11 subclones displayed a significant number of IGHV gene nucleotide differences from the dominant MM clone despite exhibiting an identical HCDR3 region. Notably, a number of these distinguishing IGHV gene mutations are silent and thus may preserve the antigenic specificity of these clonally related cells. These subclones failed our frequency criteria but the type and quality of deviations from the clonal sequence suggest that they are not artifact and warrant further investigation. Notably, all of the putative subclones were also found in the MM cDNA cohort providing further evidence as genuine subclones. The very large number of identical MM clonal sequences is consistent with the idea that MM is a post germinal center malignancy that can no longer undergo SHM. However, we present clear evidence of MM-related subclones albeit relatively small in number. The subclone sequence deviations discovered may reflect on-going genetic evolution independent of the formal SHM mechanism. Alternatively, these subclones may indeed reflect “sister” cells generated during the original germinal center reaction that resulted in the malignant clone. In summary, our studies demonstrate the extraordinary potential of this methodology to track clonal evolution in MM and its precursor conditions over time. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 470 Multiple myeloma (MM) is an incurable and fatal neoplasm characterized by the accumulation of clonal plasma cells (PC) that results in significant end organ and tissue damage. MM is preceded by either of two premalignant asymptomatic stages, monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM), both of which are identified by the presence of clonally expanded abnormal PC populations. While MGUS and SMM patients' abnormal PC populations may remain stable for years, both have a life-long significantly increased risk of progressing to MM (1% and 10% per year, respectively). For this reason, a better understanding of the molecular events prompting malignant progression and increased accuracy in identifying those patients that have begun to transition to MM is urgently needed. In this study, we identified a novel MM marker, CD147 (also known as extracellular matrix metalloproteinase inducer (MMP), or EMMPRIN), that is not only over-expressed on MM PCs as compared to its premalignant counterparts, but whose increased expression correlates with the level of abnormal PC proliferation. To our knowledge, we are the first to demonstrate a role for CD147 in MM. CD147 has been shown by others to display a variety of activities and those with potential relevance to MM include stimulation of increased MMP production and angiogenesis, and playing a critical role in glycolysis via facilitation of excess cellular lactate transport. Our initial experiments revealed that MM PCs overexpress CD147 mRNA relative to MGUS PCs. Flow cytometric analysis corroborated these data and demonstrated variable expression of CD147 across the disease continuum ranging from no expression to bimodal or uniform expression. Indeed, there was a significant difference between CD147 expression on MGUS and SMM PCs compared to that on MM PCs (p=0.02 and 0.005, respectively). We next determined whether CD147 had a signaling role in these cells. Using the natural CD147 ligand, cyclophilin B (CypB), we showed that addition of CypB to either human MM cell lines (HMCLs) or CD138+ patient PCs resulted in increased PC proliferation as measured by [3H]-thymidine incorporation. In a complementary manner, addition of CD147 antibodies significantly inhibited proliferation without an effect on cell viability. By western blot analysis we further demonstrated that CypB-mediated CD147 activation leads to MAPK phosphorylation. Next, we isolated CD147+ and CD147- MM cells from patients whose tumor cells bimodally expressed this marker and assessed the response of each subset to IL-6 and CypB. The CD147+ subset was almost solely responsible for the proliferative response in all cases examined. In addition, we cultured bone marrow mononuclear cells from CD147 bimodally expressing MM patients overnight with bromodeoxyuridine before performing cell cycle analysis on the CD147+ and CD147- MM PC populations. Remarkably, the CD147+ PCs were greatly enriched for cells in the S and G2/M phases of the cell cycle, whereas the CD147- PCs resided almost entirely in the G0/G1 phase. In the final set of experiments, we employed siRNA knockdown strategies using HMCLs to definitively test the role of CD147 in MM cell proliferation. Indeed, IL-6 induced proliferation was significantly compromised following CD147 down regulation, which was not attributed to increased apoptosis. However, IL-6 mediated phosphorylation of MAPK remained robust suggesting that the IL-6 signaling pathway overall was not compromised in these cells. Finally, cell cycle analysis demonstrated that CD147 down regulation resulted in a significant increase in the number of cells in the G0/G1 phase of the cell cycle and a decrease in the number of cells in the G2/M phase of the cell cycle, as compared to cells transfected with control siRNA. In conclusion, our data suggest that the CD147 molecule plays a critical role in the biology of malignant MM PCs, particularly as it concerns MM cell proliferation, and may thus serve as a useful and attractive target for reducing the proliferative compartment of this disease. Ongoing studies are investigating additional roles for MM cell CD147 expression, e.g., its role in MMP induction in the tumor microenviroment. Disclosures: No relevant conflicts of interest to declare.

Study Design. Retrospective matched cohort study. Objective. To determine if low-pressure lumbar provocation discography (PD) results in long-term accelerated disc degeneration, internal disc disruption, or disc herniation in patients with symptomatic low back pain (LBP). Summary of Background Data. Study of subjects without clinically-significant LBP suggests that high-pressure PD may accelerate disc degeneration. Methods. Consecutive patients with symptomatic LBP who underwent magnetic resonance imaging (MRI), PD, and repeat MRI more than 7 years later, but did not undergo subsequent spinal fusion surgery, were included. Punctured discs were matched (1:2 to 1:4) to corresponding discs in a control cohort by age, BMI, Pfirrmann score (±2), and presence of disc herniation; control cohort inclusion required MRIs for symptomatic LBP, separated by more than 7 years. The primary outcome of the study was a progression in Pfirrmann score category (I–II, III–IV, V). MRI disc-to-CSF T2 signal-intensity ratio, disc height, disc herniations, high intensity zones (HIZs), and Modic changes were assessed. Results. Baseline and follow-up MRIs were available for 77 discs exposed to PD, and for 260 discs in the matched control cohort. There was no difference in the proportion of punctured discs that advanced in Pfirrmann score category in the PD group (17%, 95% CI 9–27%) compared with corresponding discs in the Control group (21%, 95% CI 17–27%), P = 0.3578, or in non-punctured discs in the PD group (35%, 95% CI 21–51%) compared with corresponding discs in the Control group (34%, 95% CI 27–42%), P = 0.1169. There were no differences in disc-to-CSF T2 signal-intensity ratio, presence of disc herniations, HIZs, or Modic changes following puncture in the PD versus matched cohort discs or in the non-punctured PD cohort discs versus corresponding control cohort discs (P > 0.05). Conclusion. Patients with symptomatic LBP who underwent low-pressure PD, but who did not undergo a subsequent spinal fusion surgery, developed disc degeneration and new disc herniations at a similar rate to corresponding discs in matched control patients. Level of Evidence: 3

Study Design: This is a prospective cohort study. Objective: To characterize the accuracy of patient recollection of preoperative symptoms after cervical spine surgery. Summary of Background Data: Recall bias is a well-known source of systematic error. The accuracy of patient recall after cervical spine surgery remains unknown. Methods: Consecutive patients undergoing cervical spine surgery for myelopathy or radiculopathy were enrolled. Neck and arm numeric pain scores and Neck Disability Indices were recorded preoperatively. Patients were asked to recall their preoperative status at either short (<1 y) or long-term (≥1 y) follow-up. Actual and recalled scores were compared using paired t tests and relations were quantified using the Pearson correlation coefficients. Multivariable linear regression was used to identify factors impacting recollection. Results: In total, 73 patients with a mean age of 58.2 years were included. Compared with their preoperative scores, patients showed significant improvement in neck pain [mean difference (MD)=−2.9; 95% confidence intervals (CIs), −3.5 to −2.3], arm pain (MD, −3.4; 95% CI, −4.0 to −2.8), and disability (MD, −12.4%; 95% CI, −16.9 to −7.9). Patient recollection of preoperative status was significantly more severe than actual for neck pain (MD, +1.5; 95% CI, 0.8–2.2), arm pain (MD, +2.3; 95% CI, 1.6–3.0), and disability (MD, +5.8%; 95% CI, 2.4–9.2). Moderate correlation between actual and recalled scores with regard to neck (r=0.41), arm (r=0.50) pain, and disability (r=0.67) was seen. This was maintained across age, sex, and time between date of surgery and recollection. Over 30% of patients switched their predominant symptom from neck-to-arm pain or vice versa on recall of their preoperative symptoms. Conclusions: Relying on patient recollection does not provide an accurate measure of preoperative status after cervical spine surgery. Prospective and not retrospective collection of patient-reported outcomes remain the gold standard to measure and interpret outcomes after cervical spine surgery. Recall bias has the potential to affect patient satisfaction and requires further study.

ABSTRACTDegenerative disk disease of the spine is a major cause of back pain and disability. Optimization of regenerative medical therapies for degenerative disk disease requires a deep mechanistic understanding of the factors controlling the structural integrity of spinal tissues. In this investigation, we sought to identify candidate regulatory genes controlling extracellular matrix synthesis in spinal tissues. To achieve this goal we performed high throughput next generation RNA sequencing on 39 annulus fibrosus and 21 nucleus pulposus human tissue samples. Specimens were collected from patients undergoing surgical discectomy for the treatment of degenerative disk disease. Our studies identified associations between extracellular matrix genes, growth factors, and other important regulatory molecules. The fibrous matrix characteristic of annulus fibrosus was associated with expression of the growth factors platelet derived growth factor beta (PDGFB), vascular endothelial growth factor C (VEGFC), and fibroblast growth factor 9 (FGF9). Additionally we observed high expression of multiple signaling proteins involved in the NOTCH and WNT signaling cascades. Nucleus pulposus extracellular matrix related genes were associated with the expression of numerous diffusible growth factors largely associated with the transforming growth signaling cascade, including transforming factor alpha (TGFA), inhibin alpha (INHA), inhibin beta A (INHBA), bone morphogenetic proteins (BMP2, BMP6), and others. Clinical significance: this investigation provides important data on extracellular matrix gene regulatory networks in disk tissues. This information can be used to optimize pharmacologic, stem cell, and tissue engineering strategies for regeneration of the intervertebral disk and the treatment of back pain. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1356–1369, 2018.

Study Design Retrospective matched cohort analysis. Objective The majority of the literature on venous thromboembolism (VTE) after spine surgery is limited to studies of thoracolumbar surgery. Less is known regarding the incidence of VTE and associated risk factors following cervical spine surgery. Methods A total of 5,405 patients at our institution underwent cervical diskectomy, laminectomy, corpectomy, laminoplasty, or fusion between 1995 and 2012; 85 of the 5,405 patients (1.57%) suffered either a DVT (55) or pulmonary embolus (51) within 30 days postoperatively. The cases were matched 1:2 to controls based on age, sex, and date of surgery. Data regarding multiple perioperative factors, demographics, and comorbidities was collected. Results Several risk factors were identified for VTE. Significant medical comorbidities included chronic venous insufficiency (odds ratio [OR] = 3.40), atrial fibrillation (OR = 2.69), obesity (OR = 2.67), and ischemic heart disease (OR = 2.18). Staged surgery (OR = 28.0), paralysis (OR = 19.0), combined approach (OR = 7.46), surgery for infection (OR = 18.5), surgery for trauma (OR = 11.1), comorbid traumatic injuries (OR > 10), oncologic procedures (OR = 5.2), use of iliac crest autograft (OR = 4.16), two or more surgical levels (OR = 3.48), blood loss > 300 mL (OR = 1.66), and length of stay 5 days or greater (OR = 3.47) were all found to be risk factors for VTE ( p < 0.05) in univariate analysis. Multivariate analysis found staged surgery (OR = 35.7), paralysis (OR = 7.86), and nonelective surgery (OR = 6.29) to be independent risk factors for VTE. Conclusions Although the incidence of VTE following cervical spine surgery is low, we identified several risk factors that may be predictive. More aggressive approaches to prophylaxis and surveillance in certain patient populations may be warranted.

Spine surgeries tend to be lengthy (mean time of 4 hours) and highly variable (with some surgeries lasting 18 hours or more). This variability along with patient preferences driving scheduling decisions resulted in both low operating room (OR) utilization and significant overtime for surgical teams at Mayo Clinic. In this paper we discuss the development of an improved scheduling approach for spine surgeries over a rolling planning horizon. First, data mining and statistical analysis was performed using a large data set to identify categories of surgeries that could be grouped together based on surgical time distributions and could be categorized at the time of case scheduling. These surgical categories are then used in a hierarchical optimization approach with the objective of maximizing a weighted combination of OR utilization and net profit. The optimization model is explored to consider trade-offs and relationships among utilization levels, financial performance, overtime allowance, and case mix. The new scheduling approach was implemented via a custom Web-based application that allowed the surgeons and schedulers to interactively identify best surgical days with patients. A pilot implementation resulted in a utilization increase of 19% and a reduction in overtime by 10%.

Objectives To determine the incidence and 1‐year outcomes of an elderly population with perioperative atrial arrhythmia (PAA) within 7 days of hip fracture surgery. Design Retrospective cohort study. Setting The Rochester Epidemiology Project (REP). Participants Elderly adults consecutive undergoing hip fracture repair from 1988 to 2002 in Olmsted County, Minnesota (N = 1,088, mean age 84.0 ± 7.4, 80.2% female). Measurements Baseline clinical variables were analyzed in relation to survival using Cox proportional hazards methods for comparison. Results Sixty‐one participants (5.6%) developed PAA within the first 7 days. During 1 year of follow‐up, 239 (22%) participants died. PAA was associated with greater mortality (45% vs 21%; hazard ratio (HR) = 2.8, 95% confidence interval (CI) = 1.9–4.2). Other mortality risk factors were male sex (HR = 2.0, 95% CI = 1.5–2.6), congestive heart failure (HR = 2.1, 95% CI = 1.7–2.8), chronic renal insufficiency (HR = 2.0, 95% CI = 1.5–2.8), dementia (HR = 2.9, 95% CI = 2.2–3.7), and American Society of Anesthesiologists risk Class III, IV, or V (HR = 3.3, 95% CI = 1.9–5.9). Conclusion Elderly adults undergoing hip fracture surgery who develop PAA within 7 days have significantly higher 1‐year mortality than those who do not. Further studies are indicated to determine whether prevention of PAA will reduce mortality in this population.