Proteomic Portraits Reveal Evolutionarily Conserved and Divergent Responses to Spinal Cord Injury.

Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of cerebrospinal fluid and serum samples from 111 patients with acute SCI and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at 6 months with an area under the receiver operating characteristic curve of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response and establish the cerebrospinal fluid abundance of glial fibrillary acidic protein as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.

A cross-standardized flow cytometry platform to assess phenotypic stability in precursor B-cell acute lymphoblastic leukemia (B-ALL) xenografts.

With the continued poor outcome of relapsed acute lymphoblastic leukemia (ALL), new patient-specific approaches for disease progression monitoring and therapeutic intervention are urgently needed. Patient-derived xenografts (PDX) of primary ALL in immune-deficient mice have become a powerful tool for studying leukemia biology and therapy response. In PDX mice, the immunophenotype of the patient's leukemia is commonly believed to be stably propagated. In patients, however, the surface marker expression profile of the leukemic population often displays poorly understood immunophenotypic shifts during chemotherapy and ALL progression. We therefore developed a translational flow cytometry platform to study whether the patient-specific immunophenotype is faithfully recapitulated in PDX mice. To enable valid assessment of immunophenotypic stability and subpopulation complexity of the patient's leukemia after xenotransplantation, we comprehensively immunophenotyped diagnostic B-ALL from children and their matched PDX using identical, clinically standardized flow protocols and instrument settings. This cross-standardized approach ensured longitudinal stability and cross-platform comparability of marker expression intensity at high phenotyping depth. This analysis revealed readily detectable changes to the patient leukemia-associated immunophenotype (LAIP) after xenotransplantation. To further investigate the mechanism underlying these complex immunophenotypic shifts, we applied an integrated analytical approach that combined clinical phenotyping depth and high analytical sensitivity with unbiased high-dimensional algorithm-based analysis. This high-resolution analysis revealed that xenotransplantation achieves patient-specific propagation of phenotypically stable B-ALL subpopulations and that the immunophenotypic shifts observed at the level of bulk leukemia were consistent with changes in underlying subpopulation abundance. By incorporating the immunophenotypic complexity of leukemic populations, this novel cross-standardized analytical platform could greatly expand the utility of PDX for investigating ALL progression biology and assessing therapies directed at eliminating relapse-driving leukemic subpopulations.

All over the MAP: describing pressure variability in acute spinal cord injury.

STUDY DESIGN: Observational study. OBJECTIVES: To examine the feasibility of meeting the current clinical guidelines for the hemodynamic management of acute spinal cord injury (SCI) which recommend maintaining mean arterial pressure (MAP) at 85-90 mmHg in the days following injury. METHODS: This study examined data collected minute-by-minute to describe the pressure profile in the first 5 days following SCI in 16 patients admitted to the Intensive Care Unit at Vancouver General Hospital (40 ± 19 years, 13 M/3 F, C4-T11). MAP and intrathecal pressure (ITP) were monitored at 100 Hz by arterial and lumbar intrathecal catheters, respectively, and reported as the average of each minute. Spinal cord perfusion pressure was calculated as the difference between MAP and ITP. The minute-to-minute difference (MMdiff) of each pressure variable was calculated as the absolute difference between consecutive minutes. RESULTS: Only 24 ± 7% of MAP recordings were between 85 and 90 mmHg. Average MAP MMdiff was ~3 mmHg. The percentage of MAP recordings within target range was negatively correlated with the degree of variability (i.e. MMdiff; r = -0.64, p < 0.008) whereas higher mean MAP was correlated with greater variability (r = 0.57, p = 0.021). CONCLUSIONS: Our findings point to the 'real life' challenges in maintaining MAP in acute SCI patients. Given MAP fluctuated ~3 mmHg minute-to-minute, maintaining MAP within a 5 mmHg range with conventional volume replacement and vasopressors presents an almost impossible task for clinicians and warrants reconsideration of current management guidelines.

Introducing the New Patient Expectations in Spine Oncology Questionnaire.

BACKGROUND AND OBJECTIVES: It has been hypothesized that a discrepancy between pretreatment expectations and perceived outcomes is a significant source of patient dissatisfaction. Currently, there is lack in understanding and tools to assess patient expectations regarding the outcomes of treatment for spinal metastases. The objective of this study was therefore to develop a patient expectations questionnaire regarding the outcomes after surgery and/or radiotherapy for spinal metastases. METHODS: A multiphase international qualitative study was conducted. Phase 1 of the study included semistructured interviews with patients and relatives to understand their expectations of the outcomes of treatment. In addition, physicians were interviewed about their communication practices with patients regarding treatment and expected outcomes. In phase 2, items were developed based on the results of the interviews in phase 1. In phase 3, patients were interviewed to validate the content and language of the questionnaire. Selection of the final items was based on feedback from patients regarding content, language, and relevance. RESULTS: In phase 1, 24 patients and 22 physicians were included. A total of 34 items were developed for the preliminary questionnaire. After phase 3, a total of 22 items were retained for the final version of the questionnaire. The questionnaire is divided into 3 sections: (1) patient expectations regarding treatment outcomes, (2) prognosis, and (3) consultation with the physician. The items cover expectations related to pain, analgesia requirements, daily and physical function, overall quality of life, life expectancy, and information provided by the physician. CONCLUSION: The new Patient Expectations in Spine Oncology questionnaire was developed to evaluate patient expectations regarding the outcomes after treatment for spinal metastases. The Patient Expectations in Spine Oncology questionnaire will allow physicians to systematically assess patient expectations of planned treatment and thus help guide patients toward realistic expectations of treatment outcome.

Association of CSF and Serum Neurofilament Light and Glial Fibrillary Acidic Protein, Injury Severity, and Outcome in Spinal Cord Injury.

BACKGROUND AND OBJECTIVES: Traumatic spinal cord injury (SCI) is highly heterogeneous, and tools to better delineate pathophysiology and recovery are needed. Our objective was to profile the response of 2 biomarkers, neurofilament light (NF-L) and glial fibrillary acidic protein (GFAP), in the serum and CSF of patients with acute SCI to evaluate their ability to objectively characterize injury severity and predict neurologic recovery. METHODS: Blood and CSF samples were obtained from prospectively enrolled patients with acute SCI through days 1-4 postinjury, and the concentration of NF-L and GFAP was quantified using Simoa technology. Neurologic assessments defined the ASIA Impairment Scale (AIS) grade and motor score (MS) at presentation and 6 months postinjury. RESULTS: One hundred eighteen patients with acute SCI (78 AIS A, 20 AIS B, and 20 AIS C) were enrolled, with 113 (96%) completing 6-month follow-up. NF-L and GFAP levels were strongly associated between paired serum and CSF specimens, were both increased with injury severity, and distinguished among baseline AIS grades. Serum NF-L and GFAP were significantly (p = 0.02 to <0.0001) higher in AIS A patients who did not improve at 6 months, predicting AIS grade conversion with a sensitivity and specificity (95% CI) of 76% (61, 87) and 77% (55, 92) using NF-L and 72% (57, 84) and 77% (55, 92) using GFAP at 72 hours, respectively. Independent of clinical baseline assessment, a serum NF-L threshold of 170 pg/mL at 72 hours predicted those patients who would be classified as motor complete (AIS A/B) compared with motor incomplete (AIS C/D) at 6 months with a sensitivity of 87% (76, 94) and specificity of 84% (69, 94); a serum GFAP threshold of 13,180 pg/mL at 72 hours yielded a sensitivity of 90% (80, 96) and specificity of 84% (69, 94). DISCUSSION: The potential for NF-L and GFAP to classify injury severity and predict outcome after acute SCI will be useful for patient stratification and prognostication in clinical trials and inform communication of prognosis. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that higher serum NF-L and GFAP are associated with worse neurological outcome after acute SCI. TRIAL REGISTRATION INFORMATION: Registered on ClinicalTrials.gov: NCT00135278 (March 2006) and NCT01279811 (January 2012).

Characterization of Hyperacute Neuropathic Pain after Spinal Cord Injury: A Prospective Study.

There is currently a lack of information regarding neuropathic pain in the very early stages of spinal cord injury (SCI). In the present study, neuropathic pain was assessed using the Douleur Neuropathique 4 Questions (DN4) for the patient's worst pain within the first 5 days of injury (i.e., hyperacute) and on follow-up at 3, 6, and 12 months. Within the hyperacute time frame (i.e., 5 days), at- and below-level neuropathic pain were reported as the worst pain in 23% (n = 18) and 5% (n = 4) of individuals with SCI, respectively. Compared to the neuropathic pain observed in this hyperacute setting, late presenting neuropathic pain was characterized by more intense painful electrical and cold sensations, but less itching sensations. Phenotypic differences between acute and late neuropathic pain support the incorporation of timing into a mechanism-based classification of neuropathic pain after SCI. The diagnosis of acute neuropathic pain after SCI is challenged by the presence of nociceptive and neuropathic pains, with the former potentially masking the latter. This may lead to an underestimation of the incidence of neuropathic pain during the very early, hyperacute time points post-injury. TRIAL REGISTRATION: ClinicalTrials.gov (Identifier: NCT01279811) PERSPECTIVE: This article presents distinct pain phenotypes of hyperacute and late presenting neuropathic pain after spinal cord injury and highlights the challenges of pain assessments in the acute phase after injury. This information may be relevant to clinical trial design and broaden our understanding of neuropathic pain mechanisms after spinal cord injury.

Clinical and laboratory features associated with myeloperoxidase expression in pediatric B-lymphoblastic leukemia.

BACKGROUND: B-lymphoblastic leukemia (B-ALL) is the most common childhood malignancy, and its diagnosis requires immunophenotypically demonstrating blast B cell lineage differentiation. Expression of myeloperoxidase (MPO) in B-ALL is well-described and it has been recognized that a diagnosis of mixed phenotype acute leukemia should be made cautiously if MPO expression is the sole myeloid feature in these cases. We sought to determine whether MPO expression in pediatric B-ALL was associated with differences in laboratory, immunophenotypic, or clinical features. METHODS: We reviewed clinical, diagnostic bone marrow flow cytometry, and laboratory data for all new B-ALL diagnoses at our pediatric institution in 5 years. Cases were categorized as MPO positive (MPO+) or negative (MPO-) using a threshold of ≥20% blasts expressing MPO at intensity greater than the upper limit of normal lymphocytes on diagnostic bone marrow flow cytometry. RESULTS: A total of 148 cases were reviewed, 32 of which (22%) were MPO+. MPO+ B-ALL was more frequently hyperdiploid and less frequently harbored ETV6-RUNX1; no MPO+ cases had KMT2A rearrangements or BCR-ABL1. Although not significantly so, MPO+ B-ALL was less likely than MPO- B-ALL to have positive end-of-induction minimal residual disease studies (9.4 and 24%, respectively), but relapse rates and stem cell transplantation rates were similar between groups. Aberrant expression of other more typically myeloid markers was similar between these groups. CONCLUSION: In our study cohort, MPO+ B-ALL showed minimal residual disease persistence less often after induction chemotherapy but otherwise had similar clinical outcomes to MPO- B-ALL, with similar rates of additional myeloid antigen aberrancy.

Characterization of Cerebrospinal Fluid Ubiquitin C-Terminal Hydrolase L1 as a Biomarker of Human Acute Traumatic Spinal Cord Injury.

A major obstacle for translational research in acute spinal cord injury (SCI) is the lack of biomarkers that can objectively stratify injury severity and predict outcome. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a neuron-specific enzyme that shows promise as a diagnostic biomarker in traumatic brain injury (TBI), but has not been studied in SCI. In this study, cerebrospinal fluid (CSF) and serum samples were collected over the first 72-96 h post-injury from 32 acute SCI patients who were followed prospectively to determine neurological outcomes at 6 months post-injury. UCH-L1 concentration was measured using the Quanterix Simoa platform (Quanterix, Billerica, MA) and correlated to injury severity, time, and neurological recovery. We found that CSF UCH-L1 was significantly elevated by 10- to 100-fold over laminectomy controls in an injury severity- and time-dependent manner. Twenty-four-hour post-injury CSF UCH-L1 concentrations distinguished between American Spinal Injury Association Impairment Scale (AIS) A and AIS B, and AIS A and AIS C patients in the acute setting, and predicted who would remain "motor complete" (AIS A/B) at 6 months with a sensitivity of 100% and a specificity of 86%. AIS A patients who did not improve their AIS grade at 6 months post-injury were characterized by sustained elevations in CSF UCH-L1 up to 96 h. Similarly, the failure to gain >8 points on the total motor score at 6 months post-injury was associated with higher 24-h CSF UCH-L1. Unfortunately, serum UCH-L1 levels were not informative about injury severity or outcome. In conclusion, CSF UCH-L1 in acute SCI shows promise as a biomarker to reflect injury severity and predict outcome.

Empirical targets for acute hemodynamic management of individuals with spinal cord injury.

OBJECTIVE: To determine the hemodynamic conditions associated with optimal neurologic improvement in individuals with acute traumatic spinal cord injury (SCI) who had lumbar intrathecal catheters placed to measure CSF pressure (CSFP). METHODS: Ninety-two individuals with acute SCI were enrolled in this multicenter prospective observational clinical trial. We monitored mean arterial pressure (MAP) and CSFP during the first week after injury and assessed neurologic function at baseline and 6 months after injury. We used relative risk iterations to determine transition points at which the likelihood of either improving neurologically or remaining unchanged neurologically was equivalent. These transition points guided our analyses in which we examined the linear relationships between time spent within target hemodynamic ranges (i.e., clinical adherence) and neurologic recovery. RESULTS: Relative risk transition points for CSFP, MAP, and spinal cord perfusion pressure (SCPP) were linearly associated with neurologic improvement and directed the identification of key hemodynamic target ranges. Clinical adherence to the target ranges was positively and linearly related to improved neurologic outcomes. Adherence to SCPP targets, not MAP targets, was the best indicator of improved neurologic recovery, which occurred with SCPP targets of 60 to 65 mm Hg. Failing to maintain the SCPP within the target ranges was an important detrimental factor in neurologic recovery, particularly if the target range is set lower. CONCLUSION: We provide an empirical, data-driven approach to aid institutions in setting hemodynamic management targets that accept the real-life challenges of adherence to specific targets. Our results provide a framework to guide the development of widespread institutional management guidelines for acute traumatic SCI.

Predicting Injury Severity and Neurological Recovery after Acute Cervical Spinal Cord Injury: A Comparison of Cerebrospinal Fluid and Magnetic Resonance Imaging Biomarkers.

Biomarkers of acute human spinal cord injury (SCI) could provide a more objective measure of spinal cord damage and a better predictor of neurological outcome than current standardized neurological assessments. In SCI, there is growing interest in establishing biomarkers from cerebrospinal fluid (CSF) and from magnetic resonance imaging (MRI). Here, we compared the ability of CSF and MRI biomarkers to classify injury severity and predict neurological recovery in a cohort of acute cervical SCI patients. CSF samples and MRI scans from 36 acute cervical SCI patients were examined. From the CSF samples taken 24 h post-injury, the concentrations of inflammatory cytokines (interleukin [IL]-6, IL-8, monocyte chemotactic protein-1), and structural proteins (tau, glial fibrillary acidic protein, and S100ß) were measured. From the pre-operative MRI scans, we measured intramedullary lesion length, hematoma length, hematoma extent, CSF effacement, cord expansion, and maximal spinal cord compression. Baseline and 6-month post-injury assessments of American Spine Injury Association Impairment Scale (AIS) grade and motor score were conducted. Both MRI measures and CSF biomarker levels were found to correlate with baseline injury grade, and in combination they provided a stronger model for classifying baseline AIS grade than CSF or MRI biomarkers alone. For predicting neurological recovery, the inflammatory CSF biomarkers best predicted AIS grade conversion, whereas structural biomarker levels best predicted motor score improvement. A logistic regression model utilizing CSF biomarkers alone had a 91.2% accuracy at predicting AIS conversion, and was not strengthened by adding MRI features or even knowledge of the baseline AIS grade. In a direct comparison of MRI and CSF biomarkers, the CSF biomarkers discriminate better between different injury severities, and are stronger predictors of neurological recovery in terms of AIS grade and motor score improvement. These findings demonstrate the utility of measuring the acute biological responses to SCI as biomarkers of injury severity and neurological prognosis.

Spinal cord perfusion pressure predicts neurologic recovery in acute spinal cord injury.

OBJECTIVE: To determine whether spinal cord perfusion pressure (SCPP) as measured with a lumbar intrathecal catheter is a more predictive measure of neurologic outcome than the conventionally measured mean arterial pressure (MAP). METHODS: A total of 92 individuals with acute spinal cord injury were enrolled in this multicenter prospective observational clinical trial. MAP and CSF pressure (CSFP) were monitored during the first week postinjury. Neurologic impairment was assessed at baseline and at 6 months postinjury. We used logistic regression, systematic iterations of relative risk, and Cox proportional hazard models to examine hemodynamic patterns commensurate with neurologic outcome. RESULTS: We found that SCPP (odds ratio 1.039, p = 0.002) is independently associated with positive neurologic recovery. The relative risk for not recovering neurologic function continually increased as individuals were exposed to SCPP below 50 mm Hg. Individuals who improved in neurologic grade dropped below SCPP of 50 mm Hg fewer times than those who did not improve (p = 0.012). This effect was not observed for MAP or CSFP. Those who were exposed to SCPP below 50 mm Hg were less likely to improve from their baseline neurologic impairment grade (p = 0.0056). CONCLUSIONS: We demonstrate that maintaining SCPP above 50 mm Hg is a strong predictor of improved neurologic recovery following spinal cord injury. This suggests that SCPP (the difference between MAP and CSFP) can provide useful information to guide the hemodynamic management of patients with acute spinal cord injury.

A Targeted Proteomics Analysis of Cerebrospinal Fluid after Acute Human Spinal Cord Injury.

Efforts to validate novel therapies in acute clinical trials for spinal cord injury (SCI) are impeded by the lack of objective quantitative measures that reflect injury severity and accurately predict neurological recovery. Therefore, a strong rationale exists for establishing neurochemical biomarkers that objectively quantify injury severity and predict outcome. Here, we conducted a targeted proteomics analysis of cerebrospinal fluid (CSF) samples derived from 29 acute SCI patients (American Spinal Injury Association Impairment Scale [AIS] A, B, or C) acquired at 24, 48, and 72 h post-injury. From a total of 165 proteins, we identified 27 potential biomarkers of injury severity (baseline AIS A, B, or C), with triosephosphate isomerase having the strongest relationship to AIS grade. The majority of affected proteins (24 of 27) were more abundant in samples from AIS A patients than in those from AIS C patients, suggesting that for the most part, these proteins are released into the CSF more readily with more severe trauma to the spinal cord. We then analyzed the relationship between CSF protein abundance and neurological recovery. For AIS grade improvement over 6 months, we identified 34 proteins that were associated with AIS grade conversion (p < 0.05); however, these associations were not statistically significant after adjusting for multiple comparisons. For total motor score (TMS) recovery over 6 months, after adjusting for baseline neurological injury level, we identified 46 proteins with a statistically significant association with TMS recovery. Twenty-two of these proteins were among the 27 proteins that were related to baseline AIS grade, consistent with the notion that protein markers that reflect a more severe injury also appropriately predict a poorer recovery of motor function. In summary, this study provides a description of the CSF proteome changes that occur after acute human SCI, and reveals a number of protein candidates for further validation as potential biomarkers of injury severity.