Proteomics identifies novel biomarkers of synovial joint disease in a canine model of mucopolysaccharidosis I.

Mucopolysaccharidosis I is a lysosomal storage disorder characterized by deficient alpha-L-iduronidase activity, leading to abnormal accumulation of glycosaminoglycans in cells and tissues. Synovial joint disease is prevalent and significantly reduces patient quality of life. There is a critical need for improved understanding of joint disease pathophysiology in MPS I, including specific biomarkers to predict and monitor joint disease progression, and response to treatment. The objective of this study was to leverage the naturally-occurring MPS I canine model and undertake an unbiased proteomic screen to identify systemic biomarkers predictive of local joint disease in MPS I. Synovial fluid and serum samples were collected from MPS I and healthy dogs at 12 months-of-age, and protein abundance characterized using liquid chromatography tandem mass spectrometry. Stifle joints were evaluated postmortem using magnetic resonance imaging (MRI) and histology. Proteomics identified 40 proteins for which abundance was significantly correlated between serum and synovial fluid, including markers of inflammatory joint disease and lysosomal dysfunction. Elevated expression of three biomarker candidates, matrix metalloproteinase 19, inter-alpha-trypsin inhibitor heavy-chain 3 and alpha-1-microglobulin, was confirmed in MPS I cartilage, and serum abundance of these molecules was found to correlate with MRI and histological degenerative grades. The candidate biomarkers identified have the potential to improve patient care by facilitating minimally-invasive, specific assessment of joint disease progression and response to therapeutic intervention.

Imaging of Entrapment Neuropathies in the Ankle.

Entrapment neuropathies of the ankle and foot pose a major diagnostic challenge and thus remain underdiagnosed. Recent advancements in imaging modalities, including magnetic resonance neurography (MRN), have resulted in considerable improvement in the anatomical localization and identification of pathologies leading to nerve entrapment. MRN supplements clinical examination and electrophysiologic studies in the diagnosis of neuropathies, aids in assessing disease severity, and helps formulate management strategies. A comprehensive understanding of the anatomy and imaging features of the ankle is essential to diagnose and manage entrapment neuropathies accurately. Advancements in imaging and their appropriate utilization will ultimately lead to better diagnoses and improved patient outcomes.

Risk-stratifying capacity of PET/CT metabolic tumor volume in stage IIIA non-small cell lung cancer.

OBJECTIVES: Stage IIIA non-small cell lung cancer (NSCLC) is heterogeneous in tumor burden, and its treatment is variable. Whole-body metabolic tumor volume (MTVWB) has been shown to be an independent prognostic index for overall survival (OS). However, the potential of MTVWB to risk-stratify stage IIIA NSCLC has previously been unknown. If we can identify subgroups within the stage exhibiting significant OS differences using MTVWB, MTVWB may lead to adjustments in patients' risk profile evaluations and may, therefore, influence clinical decision making regarding treatment. We estimated the risk-stratifying capacity of MTVWB in stage IIIA by comparing OS of stratified stage IIIA with stage IIB and IIIB NSCLC. METHODS: We performed a retrospective review of 330 patients with clinical stage IIB, IIIA, and IIIB NSCLC diagnosed between 2004 and 2014. The patients' clinical TNM stage, initial MTVWB, and long-term survival data were collected. Patients with TNM stage IIIA disease were stratified by MTVWB. The optimal MTVWB cutoff value for stage IIIA patients was calculated using sequential log-rank tests. Univariate and multivariate cox regression analyses and Kaplan-Meier OS analysis with log-rank tests were performed. RESULTS: The optimal MTVWB cut-point was 29.2 mL for the risk-stratification of stage IIIA. We identified statistically significant differences in OS between stage IIB and IIIA patients (p < 0.01), between IIIA and IIIB patients (p < 0.01), and between the stage IIIA patients with low MTVWB (below 29.2 mL) and the stage IIIA patients with high MTVWB (above 29.2 mL) (p < 0.01). There was no OS difference between the low MTVWB stage IIIA and the cohort of stage IIB patients (p = 0.485), or between the high MTVWB stage IIIA patients and the cohort of stage IIIB patients (p = 0.459). Similar risk-stratification capacity of MTVWB was observed in a large range of cutoff values from 15 to 55 mL in stage IIIA patients. CONCLUSIONS: Using MTVWB cutoff points ranging from 15 to 55 mL with an optimal value of 29.2 mL, stage IIIA NSCLC may be effectively stratified into subgroups with no significant survival difference from stages IIB or IIIB NSCLC. This may result in more accurate survival estimation and more appropriate risk adapted treatment selection in stage IIIA NSCLC.

Targeting recruitment of disruptor of telomeric silencing 1-like (DOT1L): characterizing the interactions between DOT1L and mixed lineage leukemia (MLL) fusion proteins.

The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L865-874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia.

Requirement for Dot1l in murine postnatal hematopoiesis and leukemogenesis by MLL translocation.

Disruptor of telomeric silencing 1-like (Dot1l) is a histone 3 lysine 79 methyltransferase. Studies of constitutive Dot1l knockout mice show that Dot1l is essential for embryonic development and prenatal hematopoiesis. DOT1L also interacts with translocation partners of Mixed Lineage Leukemia (MLL) gene, which is commonly translocated in human leukemia. However, the requirement of Dot1l in postnatal hematopoiesis and leukemogenesis of MLL translocation proteins has not been conclusively shown. With a conditional Dot1l knockout mouse model, we examined the consequences of Dot1l loss in postnatal hematopoiesis and MLL translocation leukemia. Deletion of Dot1l led to pancytopenia and failure of hematopoietic homeostasis, and Dot1l-deficient cells minimally reconstituted recipient bone marrow in competitive transplantation experiments. In addition, MLL-AF9 cells required Dot1l for oncogenic transformation, whereas cells with other leukemic oncogenes, such as Hoxa9/Meis1 and E2A-HLF, did not. These findings illustrate a crucial role of Dot1l in normal hematopoiesis and leukemogenesis of specific oncogenes.

In vivo intra-articular contrast enhanced µCT imaging of mouse knee cartilage.

In vivo micro-Computed Tomography (µCT) is commonly used tool in the study of mouse bone architecture. However, in vivo imaging of mouse cartilage has been limited. Intra-articular contrast injection was evaluated for its utility in detecting mouse cartilage in µCT. Clinically used iodinated contrast agent was chosen for its widespread commercial availability. Imaging protocol was developed with wild type C57BL/6 mice for its ability to detect expected cartilage thinning that occurs with sexual maturity. The protocol was then validated with transgenic mouse model with known extracellular matrix loss. µCT findings showed good correspondence with histological assessment. In conclusion, in vivo intra-articular contrast-enhanced µCT arthrography is viable technique for evaluation of mouse cartilage. SUMMARY: In vivo intra-articular contrast enhanced µCT of the mouse knee joint can delineate cartilage thickness and extracellular matrix content. The imaging protocol may be useful for longitudinal evaluation of cartilage anomalies in transgenicmouse model.

Dose-dependent effects of enzyme replacement therapy on skeletal disease progression in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis (MPS) VII is an inherited lysosomal storage disorder characterized by deficient activity of the enzyme ß-glucuronidase. Skeletal abnormalities are common in patients and result in diminished quality of life. Enzyme replacement therapy (ERT) for MPS VII using recombinant human ß-glucuronidase (vestronidase alfa) was recently approved for use in patients; however, to date there have been no studies evaluating therapeutic efficacy in a large animal model of MPS VII. The objective of this study was to establish the effects of intravenous ERT, administered at either the standard clinical dose (4 mg/kg) or a high dose (20 mg/kg), on skeletal disease progression in MPS VII using the naturally occurring canine model. Untreated MPS VII animals exhibited progressive synovial joint and vertebral bone disease and were no longer ambulatory by age 6 months. Standard-dose ERT-treated animals exhibited modest attenuation of joint disease, but by age 6 months were no longer ambulatory. High-dose ERT-treated animals exhibited marked attenuation of joint disease, and all were still ambulatory by age 6 months. Vertebral bone disease was recalcitrant to ERT irrespective of dose. Overall, our findings indicate that ERT administered at higher doses results in significantly improved skeletal disease outcomes in MPS VII dogs.

Ultrasound guided aspiration of massive periarticular calcinosis in patient with scleroderma.

Massive periarticular calcinosis is poorly understood process arising either primarily (tumoral calcinosis) or secondary to underlying medical conditions, including connective tissue disease, soft tissue sarcoma, and metabolic dysregulation. The calcific deposits can cause functional limitation, skin ulceration, and cosmetic deformity. Treatment of the calcific deposits depends on the underlying cause but can be problematic with resistance to surgical and non-surgical treatments. Here, we introduce a case of tumoral calcinosis secondary to scleroderma treated with ultrasound guided aspiration.

Osteosarcoma Mimicking Tenosynovial Giant Cell Tumor of the Hip in a Pediatric Patient: A Case Report.

CASE: A 17-year-old boy presented to the clinic complaining of right hip pain after soccer participation. Clinical findings and imaging studies led to the diagnoses of femoroacetabular impingement and diffuse tenosynovial giant cell tumor (TGCT). Comprehensive arthroscopic management and biopsy revealed a diagnosis of osteosarcoma. The patient subsequently underwent chemotherapy, surgical resection, and reconstruction. CONCLUSION: Osteosarcoma of the proximal femur may mimic TGCT on imaging studies because osteosarcoma may show changes suggestive of inflammation. We recommend heightened clinical awareness and a comprehensive differential workup in the management of presumed TGCT about the hip in the pediatric patient population.

A Rare Incident of Intraarticular Fibromatosis of the Knee: A Case Report.

CASE: Our patient is a 34-year-old male aHthlete who presented for consultation after left knee discomfort and pressure for greater than 2 years. Advanced imaging revealed a nonspecific intraarticular suprapatellar lesion with subsequent ultrasound-guided core biopsy demonstrating a spindle cell proliferation consistent with superficial fibromatosis. Thus, the patient underwent an open en bloc surgical resection by a fellowship-trained orthopaedic oncologist. CONCLUSION: As the first reported case of intraarticular fibromatosis of the knee, this case highlights the importance of a thoughtful approach to the management of nonspecific intraarticular lesions through a comprehensive and collaborative strategy to decrease patient morbidity and optimize outcomes.

Arthroscopic Diagnosis of Occult Posterolateral Meniscocapsular Separations: Another Hidden Lesion.

PURPOSE: The purpose of this study was to describe the surgical findings and clinical outcomes in a series of patients with occult posterolateral meniscocapsular separations diagnosed arthroscopically after a negative magnetic resonance imaging (MRI) scan. METHODS: A retrospective analysis of prospectively collected data of consecutive patients who underwent surgical arthroscopy with repair of an occult posterolateral meniscocapsular separation by 2 fellowship-trained orthopaedic sports medicine surgeons at a single institution was performed. All lesions were identified arthroscopically in the posterolateral aspect of the lateral compartment as a distinct pathologic separation between the posterolateral capsule and adjacent meniscal tissue with increased excursion on probing. Clinical examination notes, MRI scans, and operative reports were reviewed. Patient-reported outcome measures were assessed via patient questionnaire. RESULTS: A total of 6 patients were included for analysis. MRI evaluation of the lateral meniscus was unrevealing in 4 patients, suggesting a possible tear of the body of the lateral meniscus in one patient and demonstrating a parameniscal cyst abutting the anterior root of the lateral meniscus in another patient. Arthroscopic examination revealed meniscocapsular separations of the posterolateral meniscus in all 6 knees, with 2 knees demonstrating concomitant bucket-handle meniscus tears. Patient-reported outcomes were determined for 67% of study patients. The average reported International Knee Documentation Committee score was 63.8, the average Knee Outcome Survey Activities of Daily Living Scale score was reported as 63, the 12-Item Short Form Survey (SF-12) Physical score averaged 46.8 with an average SF-12 Mental score of 59.9. CONCLUSIONS: The diagnosis of occult posterolateral meniscocapsular separations (MCS) could be missed on advanced imaging, such as MRI, so arthroscopic diagnosis may be required. This study indicates that arthroscopic diagnosis and repair of occult posterolateral MCS results in good functional and clinical outcomes. LEVEL OF EVIDENCE: IV, therapeutic case series.

Preliminary Radiology Report Discordances and Patient Outcomes.

RATIONALE AND OBJECTIVES: At our institution, resident and fellow radiologists issue preliminary reports for off-hours imaging studies, which are overread by attending radiologists the next day using structured discrepancy templates. In this study, we examined the impact on patient management and outcome of studies with major discordance. MATERIALS AND METHODS: For our retrospective observational study, preliminary reports between March and June 2017 that received major discordance were identified through report text search. Electronic medical records were reviewed for patient management change and patient outcome. RESULTS: Of the 199 cases, 52 cases (26%) had management change and 119 cases (60%) did not have management change. In 25 cases (13%), the preliminary report was proven correct on subsequent management. Three cases (2%) were lost to follow-up. In only one case was adverse outcome directly related to the discordant finding. In cases with patient management change, there was higher proportion of perceptual error compared with those without management change (73% versus 59%). In 47 cases (24%), the discordant finding or diagnosis was known to the clinical team, and better history could have avoided the major change. CONCLUSION: Adverse outcome from the discordant imaging finding was low (0.5%). Major change in preliminary report could be reduced with better clinical history. Patient management change was more frequently seen with perceptual errors, placing greater emphasis on strategies to reduce them.

Cape-like Anomalous Confluence of the Long Head of the Biceps and Rotator Cuff in the Presence of a SLAP Tear: A Case Report.

CASE: A 25-year-old woman sustained an acute tear of her superior anterior-posterior labrum with arthroscopic identification of an anomalous origin of the long head of the biceps arising exclusively from the supraspinatus tendon with no attachment to the supraglenoid tubercle or superior labrum. CONCLUSION: This is the first report of a symptomatic superior labrum anterior-posterior tear in a patient with a rare anatomic confluence of the long head of the biceps to the supraspinatus. We recommend addressing the labral pathology with utilization of a standard technique because satisfactory results can be realized with isolated labral fixation to the glenoid, despite the absence of an attached biceps tendon.

The Role of Dot1l in Prenatal and Postnatal Murine Chondrocytes and Trabecular Bone.

Osteoarthritis and osteoporosis are widely prevalent and have far-reaching public health implications. There is increasing evidence that epigenetics, in particular, histone 3 lysine 79 methyltransferase DOT1L, plays an important role in the cartilage and bone biology. In this study, we evaluated the role of Dot1l in the articular cartilage, growth plate, and trabecular bone utilizing conditional KO mouse models. We generated chondrocyte-specific constitutive and inducible conditional Dot1l KO mouse lines using Col2a1-Cre and Acan-CreER systems. Prenatal deletion of Dot1l in mouse chondrocytes led to perinatal mortality, accelerated ossification, and dysregulation of Col10a1 expression. Postnatal deletion of Dot1l in mouse chondrocytes resulted in trabecular bone loss decreased extracellular matrix production, and disruption of the growth plate. In addition, pharmacological inhibition of DOT1L in a progeria mouse model partially rescued the abnormal osseous phenotype. In conclusion, Dot1l is important in maintaining the growth plate, extracellular matrix production, and trabecular bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

A Rare Triad of Giant Occipital Encephalocele with Lipomyelomeningocele, Tetralogy of Fallot, and Situs Inversus.

Giant encephalocele is an uncommon congenital anomaly with very few published reports available in the English literature. Tetralogy of Fallot associated with situs inversus is also infrequently reported. To our knowledge there are no published reports of an association between giant encephalocele and Tetralogy of Fallot. The additional finding of situs inversus results in a rare pathologic triad, not heretofore described.

MLL-AF9 and MLL-ENL alter the dynamic association of transcriptional regulators with genes critical for leukemia.

OBJECTIVE: The aim of this study was to better understand how mixed lineage leukemia (MLL) fusion proteins deregulate the expression of genes critical for leukemia. MATERIALS AND METHODS: The transforming domain of one of the most common MLL fusion partners, AF9, was immunopurified after expression in myeloblastic M1 cells, and associating proteins were identified by mass spectrometric analysis. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction was used to determine how binding of associating proteins compare across Hoxa9 and Meis1 in cell lines with and without MLL fusion proteins and how binding is altered during gene down-regulation and differentiation. RESULTS: Consistent with earlier purifications of ENL and AF4 from 293 cells, the 90 amino acid C-terminal domain of AF9 associates with many other MLL translocation partners including Enl, Af4, Laf4, Af5q31, Ell, and Af10. This complex, termed elongation assisting proteins (EAPs), also contains the RNA polymerase II C-terminal domain kinase Cdk9/Cyclin T1/T2 (pTEFb) and the histone H3 lysine 79 methyltransferase Dot1L. Myeloid cells transformed by MLL fusions show higher levels and a broader distribution of EAP components at genes critical for leukemia. Inhibition of EAP components pTEFb and Dot1l show that both contribute significantly to activation of Hoxa9 and Meis1 expression. EAP is dynamically associated with the Hoxa9 and Meis1 loci in hematopoietic cells and rapidly dissociates during induction of differentiation. In the presence of MLL fusion proteins, its dissociation is prevented. CONCLUSIONS: The findings suggest that MLL fusion proteins deregulate genes critical for leukemia by excessive recruitment and impaired dissociation of EAP from target loci.