Roles of metal ions in the selective inhibition of oncogenic variants of isocitrate dehydrogenase 1.

Cancer linked isocitrate dehydrogenase (IDH) 1 variants, notably R132H IDH1, manifest a 'gain-of-function' to reduce 2-oxoglutarate to 2-hydroxyglutarate. High-throughput screens have enabled clinically useful R132H IDH1 inhibitors, mostly allosteric binders at the dimer interface. We report investigations on roles of divalent metal ions in IDH substrate and inhibitor binding that rationalise this observation. Mg2+/Mn2+ ions enhance substrate binding to wt IDH1 and R132H IDH1, but with the former manifesting lower Mg2+/Mn2+ KMs. The isocitrate-Mg2+ complex is the preferred wt IDH1 substrate; with R132H IDH1, separate and weaker binding of 2-oxoglutarate and Mg2+ is preferred. Binding of R132H IDH1 inhibitors at the dimer interface weakens binding of active site Mg2+ complexes; their potency is affected by the Mg2+ concentration. Inhibitor selectivity for R132H IDH1 over wt IDH1 substantially arises from different stabilities of wt and R132H IDH1 substrate-Mg2+ complexes. The results reveal the importance of substrate-metal ion complexes in wt and R132H IDH1 catalysis and the basis for selective R132H IDH1 inhibition. Further studies on roles of metal ion complexes in TCA cycle and related metabolism, including from an evolutionary perspective, are of interest.

Metabolic adaptations in cancers expressing isocitrate dehydrogenase mutations.

The most frequently mutated metabolic genes in human cancer are those encoding the enzymes isocitrate dehydrogenase 1 (IDH1) and IDH2; these mutations have so far been identified in more than 20 tumor types. Since IDH mutations were first reported in glioma over a decade ago, extensive research has revealed their association with altered cellular processes. Mutations in IDH lead to a change in enzyme function, enabling efficient conversion of 2-oxoglutarate to R-2-hydroxyglutarate (R-2-HG). It is proposed that elevated cellular R-2-HG inhibits enzymes that regulate transcription and metabolism, subsequently affecting nuclear, cytoplasmic, and mitochondrial biochemistry. The significance of these biochemical changes for tumorigenesis and potential for therapeutic exploitation remains unclear. Here we comprehensively review reported direct and indirect metabolic changes linked to IDH mutations and discuss their clinical significance. We also review the metabolic effects of first-generation mutant IDH inhibitors and highlight the potential for combination treatment strategies and new metabolic targets.

Isocitrate dehydrogenase variants in cancer - Cellular consequences and therapeutic opportunities.

Abnormal metabolism is common in cancer cells and often correlates with mutations in genes encoding for enzymes involved in small-molecule metabolism. Isocitrate dehydrogenase 1 (IDH1) is the most frequently mutated metabolic gene in cancer. Cancer-associated substitutions in IDH1 and IDH2 impair wild-type production of 2-oxoglutarate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) from isocitrate and oxidised nicotinamide adenine dinucleotide phosphate (NADP+ ), and substantially promote the IDH variant catalysed conversion of 2-oxoglutarate to d-2-hydroxyglutarate (d-2HG). Elevated d-2HG is a biomarker for some cancers, and inhibition of IDH1 and IDH2 variants is being pursued as a medicinal chemistry target. We provide an overview of the types of cancer-associated IDH variants, discuss some of the proposed consequences of altered metabolism as a result of elevated d-2HG, summarise therapeutic efforts targeting IDH variants and identify areas for future research.

Anion-exchange chromatography mass spectrometry provides extensive coverage of primary metabolic pathways revealing altered metabolism in IDH1 mutant cells.

Altered central carbon metabolism is a hallmark of many diseases including diabetes, obesity, heart disease and cancer. Identifying metabolic changes will open opportunities for better understanding aetiological processes and identifying new diagnostic, prognostic, and therapeutic targets. Comprehensive and robust analysis of primary metabolic pathways in cells, tissues and bio-fluids, remains technically challenging. We report on the development and validation of a highly reproducible and robust untargeted method using anion-exchange tandem mass spectrometry (IC-MS) that enables analysis of 431 metabolites, providing detailed coverage of central carbon metabolism. We apply the method in an untargeted, discovery-driven workflow to investigate the metabolic effects of isocitrate dehydrogenase 1 (IDH1) mutations in glioblastoma cells. IC-MS provides comprehensive coverage of central metabolic pathways revealing significant elevation of 2-hydroxyglutarate and depletion of 2-oxoglutarate. Further analysis of the data reveals depletion in additional metabolites including previously unrecognised changes in lysine and tryptophan metabolism.

MRS and DTI evidence of progressive posterior cingulate cortex and corpus callosum injury in the hyper-acute phase after Traumatic Brain Injury.

The posterior cingulate cortex (PCC) and corpus callosum (CC) are susceptible to trauma, but injury often evades detection. PCC Metabolic disruption may predict CC white matter tract injury and the secondary cascade responsible for progression. While the time frame for the secondary cascade remains unclear in humans, the first 24 h (hyper-acute phase) are crucial for life-saving interventions. Objectives: To test whether Magnetic Resonance Imaging (MRI) markers are detectable in the hyper-acute phase and progress after traumatic brain injury (TBI) and whether alterations in these parameters reflect injury severity. Methods: Spectroscopic and diffusion-weighted MRI data were collected in 18 patients with TBI (within 24 h and repeated 7-15 days following injury) and 18 healthy controls (scanned once). Results: Within 24 h of TBI N-acetylaspartate was reduced (F = 11.43, p = 0.002) and choline increased (F = 10.67, p = 0.003), the latter driven by moderate-severe injury (F = 5.54, p = 0.03). Alterations in fractional anisotropy (FA) and axial diffusivity (AD) progressed between the two time-points in the splenium of the CC (p = 0.029 and p = 0.013). Gradual reductions in FA correlated with progressive increases in choline (p = 0.029). Conclusions: Metabolic disruption and structural injury can be detected within hours of trauma. Metabolic and diffusion parameters allow identification of severity and provide evidence of injury progression.

A Noninvasive Comparison Study between Human Gliomas with IDH1 and IDH2 Mutations by MR Spectroscopy.

The oncogenes that are expressed in gliomas reprogram particular pathways of glucose, amino acids, and fatty acid metabolism. Mutations in isocitrate dehydrogenase genes (IDH1/2) in diffuse gliomas are associated with abnormally high levels of 2-hydroxyglutarate (2-HG) levels. The aim of this study was to determine whether metabolic reprogramming associated with IDH mutant gliomas leads to additional ¹H MRS-detectable differences between IDH1 and IDH2 mutations, and to identify metabolites correlated with 2-HG. A total of 21 glioma patients (age= 37 ± 11, 13 males) were recruited for magnetic resonance spectroscopy (MRS) using semi-localization by adiabatic selective refocusing pulse sequence at an ultra-high-field (7T). For 20 patients, the tumor mutation subtype was confirmed by immunohistochemistry and DNA sequencing. LCModel analysis was applied for metabolite quantification. A two-sample t-test was used for metabolite comparisons between IDH1 (n = 15) and IDH2 (n = 5) mutant gliomas. The Pearson correlation coefficients between 2-HG and associated metabolites were calculated. A Bonferroni correction was applied for multiple comparison. IDH2 mutant gliomas have a higher level of 2-HG/tCho (total choline=phosphocholine+glycerylphosphorylcholine) (2.48 ± 1.01vs.0.72 ± 0.38, Pc < 0.001) and myo-Inositol/tCho (2.70 ± 0.90 vs. 1.46 ± 0.51, Pc = 0.011) compared to IDH1 mutation gliomas. Associated metabolites, myo-Inositol and glucose+taurine were correlated with 2-HG levels. These results show the improved characterization of the metabolic pathways in IDH1 and IDH2 gliomas for precision medicine.

A comparison of 2-hydroxyglutarate detection at 3 and 7 T with long-TE semi-LASER.

Abnormally high levels of the 'oncometabolite' 2-hydroxyglutarate (2-HG) occur in many grade II and III gliomas, and correlate with mutations in the genes of isocitrate dehydrogenase (IDH) isoforms. In vivo measurement of 2-HG in patients, using magnetic resonance spectroscopy (MRS), has largely been carried out at 3 T, yet signal overlap continues to pose a challenge for 2-HG detection. To combat this, several groups have proposed MRS methods at ultra-high field (≥7 T) where theoretical increases in signal-to-noise ratio and spectral resolution could improve 2-HG detection. Long echo time (long-TE) semi-localization by adiabatic selective refocusing (semi-LASER) (TE = 110 ms) is a promising method for improved 2-HG detection in vivo at either 3 or 7 T owing to the use of broad-band adiabatic localization. Using previously published semi-LASER methods at 3 and 7 T, this study directly compares the detectability of 2-HG in phantoms and in vivo across nine patients. Cramér-Rao lower bounds (CRLBs) of 2-HG fitting were found to be significantly lower at 7 T (6 ± 2%) relative to 3 T (15 ± 7%) (p = 0.0019), yet were larger at 7 T in an IDH wild-type patient. Although no increase in SNR was detected at 7 T (77 ± 26) relative to 3 T (77 ± 30), the detection of 2-HG was greatly enhanced through an improved spectral profile and increased resolution at 7 T. 7 T had a large effect on pairwise fitting correlations between γ-aminobutyric acid (GABA) and 2-HG (p = 0.004), and resulted in smaller coefficients. The increased sensitivity for 2-HG detection using long-TE acquisition at 7 T may allow for more rapid estimation of 2-HG (within a few spectral averages) together with other associated metabolic markers in glioma.

Early detection of cerebral microbleeds following traumatic brain injury using MRI in the hyper-acute phase.

BACKGROUND: Traumatic brain injury (TBI) is a leading cause of death and disability in people under 45. Advanced imaging techniques to identify injury and classify severity in the first few hours and days following trauma could improve patient stratification and aid clinical decision making. Traumatic cerebral microbleeds (TCMBs), detectable on magnetic resonance susceptibility weighted imaging (SWI), can be used as markers of long-term clinical outcome. However, the relationship between TCMBs and injury severity in the first few hours after injury, and their natural evolution, is unknown. METHODS: We obtained SWI scans in 10 healthy controls, and 13 patients scanned 3-24h following TBI and again at 7-15days. TCMBs were identified and total volume quantified for every lesion in each scan. RESULTS: TCMBs were present in 6 patients, all with more severe injury classified by GCS. No lesions were identified in patients with an initial GCS of 15. Improvement in GCS in the first 15days following injury was significantly associated with a reduction in microbleed volume over the same time-period. CONCLUSION: MRI is feasible in severely injured patients in the first 24h after trauma. Detection of TCMBs using SWI provides an objective early marker of injury severity following trauma. TCMBs revealed in this time frame, offer the potential to help determine the degree of injury, improving stratification, in order to identify patients who require admission to hospital, transfer to a specialist center, or an extended period of intubation on intensive care.

Improved localisation for 2-hydroxyglutarate detection at 3T using long-TE semi-LASER.

2-hydroxyglutarate (2-HG) has emerged as a biomarker of tumour cell IDH mutations that may enable the differential diagnosis of glioma patients. At 3 Tesla, detection of 2-HG with magnetic resonance spectroscopy is challenging because of metabolite signal overlap and a spectral pattern modulated by slice selection and chemical shift displacement. Using density matrix simulations and phantom experiments, an optimised semi-LASER scheme (TE = 110 ms) improves localisation of the 2-HG spin system considerably compared to an existing PRESS sequence. This results in a visible 2-HG peak in the in vivo spectra at 1.9 ppm in the majority of IDH mutated tumours. Detected concentrations of 2-HG were similar using both sequences, although the use of semi-LASER generated narrower confidence intervals. Signal overlap with glutamate and glutamine, as measured by pairwise fitting correlation was reduced. Lactate was readily detectable across glioma patients using the method presented here (mean CLRB: (10±2)%). Together with more robust 2-HG detection, long TE semi-LASER offers the potential to investigate tumour metabolism and stratify patients in vivo at 3T.

Optimising treatment strategies in spinal ependymoma based on 20years of experience at a single centre.

Spinal ependymomas are rare tumours, with total resection favoured where possible. Several case series assessing the outcome following neurosurgical treatment for spinal ependymoma advocate the usage of adjuvant radiotherapy in cases of subtotal resection, or in unencapsulated tumours. We assessed the outcome of 61 consecutive cases of spinal ependymoma in a single centre over a 20year period using a variety of outcome measures. Sex distribution was equal, with a mean age at surgery of 43.6years (range 5-76years). Overall, most tumours occurred in the lumbosacral region (70.5%), with fewer in the thoracic (27.9%) and cervical regions (18.0%). Myxopapillary features were seen in 41.0% of tumours, and were more common when occurring in the lumbar region (51.2%). Gross total resection was achieved in 52.5%, subtotal resection in 37.7% and biopsy alone in 9.8% of patients and 31.1% received adjuvant radiotherapy. Two-thirds of patients achieved an excellent post-operative neurological outcome (Frankel grade E). Tumour recurrence was rare. Gross total resection and good preoperative neurological condition were most strongly predictive of good outcome. Post-operative radiotherapy did not seem to confer survival benefit in this case series, even in cases of incomplete resection, leading us to question its utility for all cases of spinal cord ependymoma.

Noninvasive Quantification of 2-Hydroxyglutarate in Human Gliomas with IDH1 and IDH2 Mutations.

Mutations in the isocitrate dehydrogenase genes (IDH1/2) occur often in diffuse gliomas, where they are associated with abnormal accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). Monitoring 2-HG levels could provide prognostic information in this disease, but detection strategies that are noninvasive and sufficiently quantitative have yet to be developed. In this study, we address this need by presenting a proton magnetic resonance spectroscopy ((1)H-MRS) acquisition scheme that uses an ultrahigh magnetic field (≥ 7T) capable of noninvasively detecting 2-HG with quantitative measurements sufficient to differentiate mutant cytosolic IDH1 and mitochondrial IDH2 in human brain tumors. Untargeted metabolomics analysis of in vivo (1)H-MRS spectra discriminated between IDH-mutant tumors and healthy tissue, and separated IDH1 from IDH2 mutations. High-quality spectra enabled the quantification of neurochemical profiles consisting of at least eight metabolites, including 2-HG, glutamate, lactate, and glutathione in both tumor and healthy tissue voxels. Notably, IDH2 mutation produced more 2-HG than IDH1 mutation, consistent with previous findings in cell culture. By offering enhanced sensitivity and specificity, this scheme can quantitatively detect 2-HG and associated metabolites that may accumulate during tumor progression, with implications to better monitor patient responses to therapy.

Idiopathic normal pressure hydrocephalus: an important differential diagnosis.

Idiopathic normal pressure hydrocephalus, an uncommon but important differential diagnosis for ataxia, cognitive impairment and urinary incontinence, is surgically treatable, unlike many of its differential diagnoses. This article discusses its assessment, investigation and therapeutic interventions.

Anterior cervical discectomy plus intervertebral polyetheretherketone cage fusion over three and four levels without plating is safe and effective long-term.

Anterior cervical discectomy and fusion (ACDF) is an established treatment for single-level cervical spondylotic myelopathy and radiculopathy, yet its stand-alone use for multi-level disease of the subaxial cervical spine remains controversial. We report a prospectively studied case series of 30 patients receiving polyetheretherketone (PEEK) cage fusion over three and four cervical levels without anterior plating. Seven (23.3%) four-level procedures (all C3 to C7) were performed, the other 23 (76.7%) being three-level, with 19 (64.4%) at C4 to C7 and four (12.3%) at C3 to C6. Long-term follow-up of more than 2 years was available in 67% of patients. This cohort showed statistically significant improvements in visual analogue score for neck pain (p=0.0006), arm pain (p=0.0003) and Japanese Orthopaedic Association myelopathy score (p=0.002). Fused segment heights increased by 0.6-1.1%. Adjacent segment disease requiring ACDF at C3-4 was seen in 6.7% of patients (one after trauma) at a mean follow-up of 62 months. Same segment recurrence requiring posterior decompression with instrumented fusion was found in 10% of patients at a mean follow-up of 49 months, only one of whom had radiological evidence of cage subsidence. The results suggest the procedure is safe and effective with potentially less morbidity than anterior plating, shorter inpatient stays than posterior approaches, acceptable same segment recurrence and lower than predicted adjacent segment disease rates.

Use of the Solis cage and local autologous bone graft for anterior cervical discectomy and fusion: early technical experience.

OBJECT: The authors prospectively evaluated the clinical and radiological outcomes after anterior cervical discectomy and fusion (ACDF) involving placement of a Solis cage and local autograft in patients who presented with symptomatic cervical spondylosis. METHODS: Twenty-two consecutive patients underwent ACDF for radiculopathy (13 cases), myeloradiculopathy (eight cases), or myelopathy alone (one case) and were assessed at 3, 6, and 12 months. Plain cervical spine radiography demonstrated a significant change in both local (p < 0.05) and regional (p < 0.05) kyphotic angles as well as an increase in segmental height (p < 0.05). At 12 months, plain radiography demonstrated Grades I, II, and III new bone formation in two, three, and 17 patients, respectively. Clinical outcomes were assessed using a visual analog scale for both neck and arm pain and a modified Japanese Orthopaedic Association (JOA) scale for myelopathy. There was a significant improvement in both arm (p < 0.05) and neck pain (p < 0.05). At 12 months, 16 (84%) of 19 and 19 (86%) of 22 patients reported complete resolution of arm pain and neck pain, respectively. There was a significant improvement in JOA scores following surgery (p < 0.05). There were two complications in the series: one case of deep venous thrombosis and one case of postoperative arm pain that resolved after conservative treatment. There were no technical complications. CONCLUSIONS: Early experience with Solis cage-augmented ACDF indicates good clinical and radiological outcomes; additionally, there are the advantages of absent donor site morbidity and anterior plate system-related morbidity.

The clinical syndrome associated with lumbar spinal stenosis.

Lumbar spinal stenosis is well defined in patho-anatomical terms but its clinical features are heterogeneous. We carried out a comprehensive retrospective review of the clinical features, radiological changes and outcome of 75 patients with radiologically diagnosed lumbar spinal stenosis in order to define its clinical spectrum. The presenting complaints were of weakness, numbness/tingling, radicular pain and neurogenic claudication in almost equal proportions. The commonest symptom was numbness or tingling of the legs. Neurogenic claudication eventually occurred in only 61%. Ninety-three per cent showed abnormalities on neurological examination, but these were generally mild with reduced ankle jerks being commonest. Imaging of the lumbar spine showed that moderate to severe central spinal stenosis correlated with complaints of weakness and abnormal motor power on clinical examination. Patients were reviewed at a mean of 4 years after diagnosis and 65% had undergone surgical decompression; this was not a prospective comparison of different treatment modalities. Overall, a third of patients felt that their symptoms had improved while a quarter felt that they had worsened. More than half had satisfactory neurological function at the time of review. Thirty-nine per cent of those treated surgically, and 25% of those managed conservatively, reported improved symptoms. A poorer functional status at review correlated with complaints of motor weakness and associated comorbid disease. Degenerative lumbar stenosis is a clinically heterogeneous neurological disorder of the lower limbs in the elderly with variable longer-term outcome. A high index of suspicion is required and neuroimaging should be obtained to confirm the diagnosis.

Increase in apparent diffusion coefficient in normal appearing white matter following human traumatic brain injury correlates with injury severity.

Following diffuse traumatic brain injury, there may be persistent functional or psychological deficits despite the presence of normal conventional MR images. Previous experimental animal and human studies have shown diffusion abnormalities following focal brain injury. Our aim was to quantify changes in apparent diffusion coefficient (ADC) and absolute relaxation times of normal appearing white matter (NAWM) in humans following traumatic brain injury. Twenty-three patients admitted with a diagnosis of head injury (nine mild, eight moderate, and six severe) were scanned an average of 7.6 days after injury using a quantitative echo planar imaging acquisition to obtain co-registered T1, T2, and ADC parametric maps. Mean NAWM values were compared with a control group (n = 13). The patient group showed a small but significant increase in ADC in NAWM, with no significant change in T1 or T2 relaxation times. There was a correlation between injury severity and increasing ADC (p = 0.03) but no correlation with either T1 or T2, suggesting that ADC is a sensitive and independent marker of diffuse white matter tissue damage following traumatic insult. None of the patients had a reduced ADC, making ischaemia unlikely in this cohort. Pathophysiological mechanisms that may explain diffusely raised ADC include vasogenic edema, chronic ischemic phenomena, or changes in tissue cytoarchitecture or neurofilament alignment.

Anterior correction of cervical kyphotic deformity: effects on myelopathy, neck pain, and sagittal alignment.

OBJECT: Cervical myelopathy may develop as a result of spinal cord compression with or without deformity. The effect of persistent kyphotic deformity on the ability of the cervical cord to recover following decompressive surgery is not known. METHODS: Between 1997 and 2000, a total of 28 patients with progressive myelopathy and kyphotic deformity underwent anterior decompression, deformity correction (0-4 degrees of lordosis), and fusion with anterior plating. Patients received clinical and radiological follow-up care, with independent analysis. Variables assessed included patient characteristics, severity of preoperative myelopathy, neck pain, and cervical sagittal alignment. Twenty-six patients (93%) underwent follow-up review for a minimum of 18 months. Two patients died: one died in the perioperative period and was excluded from further analysis, and in the other only 3 months of follow-up data could be obtained. Local deformity was corrected to neutral or lordosis in 24 cases (89%), and the overall cervical curve was corrected to neutral or lordosis in 20 cases (74%). There was a significant improvement in myelopathy scores in those patients in whom the target (0 to 4 degrees of lordosis) local angle was achieved (p = 0.04). There was a variable change in overall cervical sagittal alignment following local correction. Improvement in myelopathy was unrelated to patient age, previous surgery, or number of segments fused. Improvement in pain score was not related to correction of kyphotic angle. CONCLUSIONS: The correction of sagittal alignment may promote recovery in spinal cord function in patients with kyphotic deformity.