Discovery of Modified Metabolites, Secondary Metabolites, and Xenobiotics by Structure-Oriented LC-MS/MS.

Exogenous compounds and metabolites derived from therapeutics, microbiota, or environmental exposures directly interact with endogenous metabolic pathways, influencing disease pathogenesis and modulating outcomes of clinical interventions. With few spectral library references, the identification of covalently modified biomolecules, secondary metabolites, and xenobiotics is a challenging task using global metabolomics profiling approaches. Numerous liquid chromatography-coupled mass spectrometry (LC-MS) small molecule analytical workflows have been developed to curate global profiling experiments for specific compound groups of interest. These workflows exploit shared structural moiety, functional groups, or elemental composition to discover novel and undescribed compounds through nontargeted small molecule discovery pipelines. This Review introduces the concept of structure-oriented LC-MS discovery methodology and aims to highlight common approaches employed for the detection and characterization of covalently modified biomolecules, secondary metabolites, and xenobiotics. These approaches represent a combination of instrument-dependent and computational techniques to rapidly curate global profiling experiments to detect putative ions of interest based on fragmentation patterns, predictable phase I or phase II metabolic transformations, or rare elemental composition. Application of these methods is explored for the detection and identification of novel and undescribed biomolecules relevant to the fields of toxicology, pharmacology, and drug discovery. Continued advances in these methods expand the capacity for selective compound discovery and characterization that promise remarkable insights into the molecular interactions of exogenous chemicals with host biochemical pathways.

Point-of-care ultrasound in geriatrics: a national survey of VA medical centers.

BACKGROUND: Point-of-care ultrasound (POCUS) can aid geriatricians in caring for complex, older patients. Currently, there is limited literature on POCUS use by geriatricians. We conducted a national survey to assess current POCUS use, training desired, and barriers among Geriatrics and Extended Care ("geriatric") clinics at Veterans Affairs Medical Centers (VAMCs). METHODS: We conducted a prospective observational study of all VAMCs between August 2019 and March 2020 using a web-based survey sent to all VAMC Chiefs of Staff and Chiefs of geriatric clinics. RESULTS: All Chiefs of Staff (n=130) completed the survey (100% response rate). Chiefs of geriatric clinics ("chiefs") at 76 VAMCs were surveyed and 52 completed the survey (68% response rate). Geriatric clinics were located throughout the United States, mostly at high-complexity, urban VAMCs. Only 15% of chiefs responded that there was some POCUS usage in their geriatric clinic, but more than 60% of chiefs would support the implementation of POCUS use. The most common POCUS applications used in geriatric clinics were the evaluation of the bladder and urinary obstruction. Barriers to POCUS use included a lack of trained providers (56%), ultrasound equipment (50%), and funding for training (35%). Additionally, chiefs reported time utilization, clinical indications, and low patient census as barriers. CONCLUSIONS: POCUS has several potential applications for clinicians caring for geriatric patients. Though only 15% of geriatric clinics at VAMCs currently use POCUS, most geriatric chiefs would support implementing POCUS use as a diagnostic tool. The greatest barriers to POCUS implementation in geriatric clinics were a lack of training and ultrasound equipment. Addressing these barriers systematically can facilitate implementation of POCUS use into practice and permit assessment of the impact of POCUS on geriatric care in the future.

Identification of Formaldehyde-Induced DNA-RNA Cross-Links in the A/J Mouse Lung Tumorigenesis Model.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco products, and exposure to it is likely one of the factors contributing to the development of lung cancer in cigarette smokers. To exert its carcinogenic effects, NNK must be metabolically activated into highly reactive species generating a wide spectrum of DNA damage. We have identified a new class of DNA adducts, DNA-RNA cross-links found for the first time in NNK-treated mice lung DNA using our improved high-resolution accurate mass segmented full scan data-dependent neutral loss MS3 screening strategy. The levels of these DNA-RNA cross-links were found to be significantly higher in NNK-treated mice compared to the corresponding controls, which is consistent with higher levels of formaldehyde due to NNK metabolism as compared to endogenous levels. We hypothesize that this DNA-RNA cross-linking occurs through reaction with NNK-generated formaldehyde and speculate that this phenomenon has broad implications for NNK-induced carcinogenesis. The structures of these cross-links were characterized using high-resolution LC-MS2 and LC-MS3 accurate mass spectral analysis and comparison to a newly synthesized standard. Taken together, our data demonstrate a previously unknown link between DNA-RNA cross-link adducts and NNK and provide a unique opportunity to further investigate how these novel NNK-derived DNA-RNA cross-links contribute to carcinogenesis in the future.

Childhood Systemic Lupus Erythematosus: Presentation, management and long-term outcomes in an Australian cohort.

OBJECTIVES: Systemic Lupus Erythematosus (SLE) is a serious autoimmune disease often resulting in major end-organ damage and increased mortality. Currently, no data exists focussing on the presentation, long-term management and progression of SLE in the Australian paediatric population. We conducted the first Australian longitudinal review of childhood SLE, focussing on response to treatment and outcomes. METHODS: Detailed clinical and laboratory data of 42 children diagnosed with SLE before 16 years from 1998 to 2018 resident in Western Australia was collected. Data was collected at diagnosis and key clinical review time points and compared using the Systemic Lupus Collaborating Clinics (SLICC) and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) criteria. End organ damage was assessed against Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI). Incidence rates of disease complications and end organ damage were determined. RESULTS: Of the 42 children, 88% were female with average age at diagnosis of 12.5 years. Indigenous Australians were over represented with an incidence rate 18-fold higher than non-Indigenous, although most children were Caucasian, reflecting the demographics of the Australian population. Median duration of follow-up was 4.25 years. On final review, 28.6% had developed cumulative organ damage as described by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (incidence rate: 0.08/PY (95% CI 0.04-0.14)), and one child died. Twenty-nine children had renal involvement (incidence rate: 0.38/PY (95% CI 0.26-0.56)). Of the 27 patients with biopsy proven lupus nephritis, 70% had Class III or IV disease. Average length of prednisolone use from diagnosis was 32.5 months. Hydroxychloroquine (n = 36) and mycophenolate mofetil (n =21) were the most widely used steroid sparing agents. 61.9% received rituximab and/or cyclophosphamide. CONCLUSION: This is the first longitudinal retrospective review of Australian children with SLE, with a markedly higher incidence in Indigenous children. Although improving, rates of end organ complications remain high, similar to international cohort outcomes. Longitudinal multi-centre research is crucial to elucidate risk factors for poor outcomes, and identifying those warranting early more aggressive therapy.

Epidemiology of Kawasaki disease in Australia using two nationally complete datasets.

AIM: The incidence of Kawasaki disease (KD) is reported to be increasing in some populations. We sought to describe long-term trends in the incidence and epidemiology of KD in Australia over 25 years. METHODS: Two nationally complete administrative datasets relevant to KD in Australia were analysed and compared. The Australian Red Cross Lifeblood Supply Tracking Analysis Reporting System (STARS) recorded all doses of immunoglobulin (IVIG) approved in Australia between January 2007 and June 2016. The Australian Institute of Health and Welfare National Hospital Morbidity Database (NHMD) records all episodes of care in hospitals across Australia. Data relevant to KD were extracted an analysed, with comparisons made for the period of data overlap. RESULTS: During the period of data overlap (2007-2015) the IVIG treatment rate in the 0- to 4-year age group (calculated from STARS) was 14.31 per 100 000 person-years (95% confidence interval 13.67-14.97). The hospitalisation rate in the same age group (calculated from the NHMD) was 14.99 per 100 000 person-years (95% confidence interval 14.33-15.66). Hospitalisation rates rose at an average rate of 3.54% annually over the 25 years to 2017 in the 0- to 4-year age group, almost exclusively in the 1- to 4-year age group. CONCLUSIONS: There is evidence of increasing KD diagnosis in Australia. Similar trends have also been reported in Asia but not in North America or Europe. Increasing diagnosis may reflect a true increase in disease incidence, increasing recognition or overdiagnosis. Further research is needed to determine the cause for these trends.

Extension of Diagnostic Fragmentation Filtering for Automated Discovery in DNA Adductomics.

Development of high-resolution/accurate mass liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) methodology enables the characterization of covalently modified DNA induced by interaction with genotoxic agents in complex biological samples. Constant neutral loss monitoring of 2'-deoxyribose or the nucleobases using data-dependent acquisition represents a powerful approach for the unbiased detection of DNA modifications (adducts). The lack of available bioinformatics tools necessitates manual processing of acquired spectral data and hampers high throughput application of these techniques. To address this limitation, we present an automated workflow for the detection and curation of putative DNA adducts by using diagnostic fragmentation filtering of LC-MS/MS experiments within the open-source software MZmine. The workflow utilizes a new feature detection algorithm, DFBuilder, which employs diagnostic fragmentation filtering using a user-defined list of fragmentation patterns to reproducibly generate feature lists for precursor ions of interest. The DFBuilder feature detection approach readily fits into a complete small-molecule discovery workflow and drastically reduces the processing time associated with analyzing DNA adductomics results. We validate our workflow using a mixture of authentic DNA adduct standards and demonstrate the effectiveness of our approach by reproducing and expanding the results of a previously published study of colibactin-induced DNA adducts. The reported workflow serves as a technique to assess the diagnostic potential of novel fragmentation pattern combinations for the unbiased detection of chemical classes of interest.

High-throughput Identification of FLT3 Wild-type and Mutant Kinase Substrate Preferences and Application to Design of Sensitive In Vitro Kinase Assay Substrates.

Acute myeloid leukemia (AML) is an aggressive disease that is characterized by abnormal increase of immature myeloblasts in blood and bone marrow. The FLT3 receptor tyrosine kinase plays an integral role in hematopoiesis, and one third of AML diagnoses exhibit gain-of-function mutations in FLT3, with the juxtamembrane domain internal tandem duplication (ITD) and the kinase domain D835Y variants observed most frequently. Few FLT3 substrates or phosphorylation sites are known, which limits insight into FLT3's substrate preferences and makes assay design particularly challenging. We applied in vitro phosphorylation of a cell lysate digest (adaptation of the Kinase Assay Linked with Phosphoproteomics (KALIP) technique and similar methods) for high-throughput identification of substrates for three FLT3 variants (wild-type, ITD mutant, and D835Y mutant). Incorporation of identified substrate sequences as input into the KINATEST-ID substrate preference analysis and assay development pipeline facilitated the design of several peptide substrates that are phosphorylated efficiently by all three FLT3 kinase variants. These substrates could be used in assays to identify new FLT3 inhibitors that overcome resistant mutations to improve FLT3-positive AML treatment.

Hypermobility and Musculoskeletal Pain in Adolescents.

OBJECTIVE: To determine the prevalence of generalized joint hypermobility (GJH) in a large cohort of Australian children and determine the associations between GJH and musculoskeletal pain. STUDY DESIGN: This is a cross-sectional analysis of the Western Australian Pregnancy Cohort (Raine) Study. Hypermobility was measured in 1584 participants at 14 years of age using the Beighton scoring system, along with a range of other factors including musculoskeletal pain status. Logistic regression models were used to assess independent associations of GJH with factors of interest. RESULTS: The prevalence of GJH was 60.6% and 36.7% in girls and boys, respectively, when defined as a Beighton score of ≥4; when defined as ≥6, it was 26.1% and 11.5%. In girls, positive associations between GJH and higher socioeconomic status and better motor competence were observed. In boys, positive associations between GJH and lower body mass index were observed. After adjusting for potential confounders, an association between number of pain areas in the last month and made worse with sport were identified in boys but not girls. CONCLUSION: The high prevalence rates of GJH as defined by commonly used Beighton cutoff values in this cohort highlight the need to question the appropriateness of these cutoffs in future studies. Future prospective studies of the association between GJH and musculoskeletal pain should be adjusted for confounding variables identified in this study, and be powered for sex-specific analyses owing to the differing prevalence rates and hypermobility correlates in male and female samples.

Chronic recurrent multifocal osteomyelitis in children: nine years' experience at a statewide tertiary paediatric rheumatology referral centre.

OBJECTIVE: To describe the clinical features, management and outcome of 34 children with chronic recurrent multifocal osteomyelitis (CRMO) diagnosed at a single centre over 9 years. METHODS: All children identified with CRMO for the period 2005-13 were identified from a prospectively collected database, with additional data from hospital records. RESULTS: Thirty-four patients, 21 female and 13 male, were identified. The average age at symptom onset was 9.8 years (range 3.8-17.9) and at diagnosis was 10.9 years (range 5.2-18.2), with an average delay in diagnosis of 12 months. Follow-up was 0.3-7.9 years (average 2.1), with 104 individual bony lesions identified, with a median of 3 (range 1-9) per patient. Six patients had unifocal disease. The sites involved included the tibia (n = 19), femur (n = 14), clavicle (n = 12), vertebrae (n = 10) and fibula (n = 8). Approximately half of patients had an inflammatory arthritis at diagnosis, and two-thirds in total eventually developed an arthritis. Pustulosis occurred in eight patients (24%), severe acne in four (12%) and psoriasis in three (9%). NSAIDs were used in 91%, CSs in 82% and MTX in 38%. Two patients were treated with anti-TNF agents. Episodic disease was most common (79%), while 21% had a monophasic pattern. Clinical remission occurred in 94% of children, with prolonged remission in 17%. Seven patients did not require medications for >12 months. CONCLUSION: CRMO is more common than previously recognized, but diagnosis may be delayed. Episodic multifocal disease was most common, but some had unifocal and/or monophasic disease. Most patients responded to NSAIDs and/or intermittent CSs, but many required DMARDs.

Current Use and Barriers to Point-of-Care Ultrasound in Rheumatology: A National Survey of VA Medical Centers.

INTRODUCTION: Point-of-care ultrasound (POCUS) can assist rheumatologists in monitoring disease activity, establishing diagnoses, and guiding procedural interventions. POCUS use has been increasing, but little is known about current use and barriers among rheumatologists. The purpose of this study was to characterize current POCUS use, training needs, and barriers to use among rheumatologists in practice. METHODS: A prospective observational study of all Veterans Affairs (VA) medical centers was conducted using a web-based survey sent to all chiefs of staff and rheumatology chiefs about current POCUS use, training needs, barriers, and policies. RESULTS: All chiefs of staff (n = 130) and rheumatology chiefs at VA medical centers (n = 95) were surveyed with 100% and 84% response rates, respectively. The most common diagnostic POCUS applications were evaluation of synovitis, joint effusion, tendinopathies, bursitis, and rotator cuff. The most common procedural applications were arthrocentesis and joint, bursa, and tendon injection. Most rheumatology chiefs (69%) expressed interest in training for their group. The most common barriers to POCUS use were lack of trained providers (68%), funding for training (54%), training opportunities (38%), funding for travel (38%), and ultrasound equipment (31%). Lack of POCUS infrastructure was common, and few facilities had POCUS policies (20%), image archiving (25%), or quality assurance processes (6%). CONCLUSION: Currently, half of rheumatology groups use diagnostic and procedural ultrasound applications. Most rheumatology groups desire training, and lack of training and equipment were the most common barriers to ultrasound use. Deliberate investment is needed in ultrasound training and infrastructure for systematic adoption of POCUS in rheumatology. Graphical Abstract available for this article. TRIAL REGISTRATION: NCT03296280.

Major cluster of chilblain cases in a cold dry Western Australian winter.

AIM: Primary chilblains are an idiopathic cold-induced vasculopathy affecting the soft tissues of the hands and feet. Secondary chilblains occur in different forms of vasculitis and chronic autoimmune connective tissue disorders. Idiopathic chilblains are rarely reported in children and may generate significant anxiety to doctors and patients. We describe a cluster of idiopathic chilblains encountered over the winter of 2010 in Perth, Western Australia. METHODS: This is a retrospective review of patients identified from a prospectively compiled database of all new cases seen in our department. Data on history, examination, investigations, prescribed treatments and outcomes were collected. RESULTS: Thirty-two patients with isolated idiopathic chilblains were included, including 20 females and 12 males with a median age at onset of 13.5 years. Lesions were papular with signs of peripheral vasoconstriction causing acrocyanosis, and uncomfortable due to pain and/or pruritis in most. Thickening of the small joints was common where lesions involved these areas. Ulceration of lesions also occurred in some. One patient required hospitalisation for secondary bacterial infection. Most received some form of treatment including non-steroidal anti-inflammatory drugs, prednisolone or nifedipine. Most patients improved spontaneously with warmer weather or responded to cold protection advice. All had resolved completely by late spring (November). CONCLUSION: Our cluster of chilblains was associated with an unusually cold winter in Perth 2010. It is the largest series reported in the literature, suggesting that chilblains may be more common than previously thought. Chilblains are almost always benign in nature and patients are systemically well and usually need no further investigation and only symptomatic treatment. Prompt recognition can avoid excessive investigation and anxiety, allowing appropriate simple advice and treatment.

Use of bisphosphonates for the treatment of osteonecrosis as a complication of therapy for childhood acute lymphoblastic leukaemia (ALL).

BACKGROUND: Osteonecrosis is a well-recognised complication of current childhood acute lymphoblastic leukaemia (ALL) therapy. There are few studies on the medical management of osteonecrosis in this setting. We studied the therapeutic and radiological effects of oral and intravenous bisphosphonate use compared with standard care as treatment for osteonecrosis in this population. METHOD: Patients who developed osteonecrosis as a complication of ALL therapy between 1994 and 2007 were treated at a single paediatric institution. Of 17 patients, 9 were commenced on bisphosphonates and 8 treated conservatively. Both groups were observed with time. Pain, analgesic requirement and musculoskeletal function were assessed monthly. Affected joints were radiologically imaged at set intervals. Each scan was graded using an ellipsoid method to give the total volume of osteonecrosis, by blinded radiologic examination. RESULTS: Three of six patients treated with oral alendronate showed clinical improvement. The three patients that had no improvement were subsequently treated with intravenous pamidronate. All six patients treated with intravenous pamidronate showed clinical improvement. Seven of eight conservatively treated patients deteriorated clinically. All patients demonstrated reduction in the radiological burden of osteonecrosis with time. There was no difference in the rate of reduction between conservative and bisphosphonate arms. CONCLUSION: Bisphosphonate use, in particular pamidronate, improved pain scores, analgesic requirement and musculoskeletal function in patients with osteonecrosis occurring as a complication of childhood ALL therapy. Objective radiologic benefit of bisphosphonate treatment could not be demonstrated. Risks, benefits and long-term outcome of bisphosphonate use in this population should be addressed in a larger prospective, randomised trial.

Two subtypes of symptomatic joint hypermobility: a descriptive study using latent class analysis.

OBJECTIVE: To investigate a cohort of children with symptomatic joint hypermobility. METHODS: Case notes for 318 children with joint hypermobility attending a rheumatology clinic were reviewed for clinical presentation, medical history, psychosocial factors and physical examination findings. Seven key variables were extracted and used as indicator variables in a latent class analysis to estimate the presence and number of subgroups of children with symptomatic joint hypermobility. RESULTS: Two subgroups with differing clinical presentations were identified accounting for age and gender: an 'athletic-persistent' class (62%) characterised by higher probabilities for recurrent and chronic musculoskeletal pain, and less severe hypermobility; and a 'systemic-profound' class (38%) characterised by generalised hypermobility, recurrent musculoskeletal pain, gastro-oesophageal reflux and motor delay. CONCLUSION: Findings suggest the presence of two distinct presentations of children with hypermobility. This finding may be important for clinical decision-making and management of this group of children.

Metabolic perturbations in Welsh Ponies with insulin dysregulation, obesity, and laminitis.

BACKGROUND: Metabolomics, the study of small-molecule metabolites, has increased understanding of human metabolic diseases, but has not been used to study equine metabolic syndrome (EMS). OBJECTIVES: (1) To examine the serum metabolome of Welsh Ponies with and without insulin dysregulation before and during an oral sugar test (OST). (2) To identify differences in metabolites in ponies with insulin dysregulation, obesity, or history of laminitis. ANIMALS: Twenty Welsh Ponies (mean ± SD; 13.8 ± 9.0 years) classified as non-insulin dysregulated [CON] (n = 10, insulin < 30 mU/L) or insulin dysregulated [ID] (n = 10, insulin > 60 mU/L) at 75 minutes after administration of Karo syrup, obese (n = 6) or nonobese (n = 14), and history of laminitis (n = 9) or no history of laminitis (n = 11). METHODS: Case-control study. Metabolomic analysis was performed on serum obtained at 0 minutes (baseline) and 75 minutes during the OST. Data were analyzed with multivariable mixed linear models with significance set at P ≤ .05. RESULTS: Metabolomic analysis of 646 metabolites (506 known) detected significant metabolite differences. At baseline, 55 metabolites (insulin response), 91 metabolites (obesity status), and 136 metabolites (laminitis history) were different. At 75 minutes, 51 metabolites (insulin response), 102 metabolites (obesity status), and 124 metabolites (laminitis history) were different. CONCLUSIONS AND CLINICAL IMPORTANCE: Use of metabolomics could have diagnostic utility for early detection of EMS and provide new knowledge regarding the pathophysiology of metabolic perturbations associated with this condition that might lead to improved clinical management.

Temporomandibular Joint Involvement in Association With Quality of Life, Disability, and High Disease Activity in Juvenile Idiopathic Arthritis.

OBJECTIVE: To evaluate the demographic, disease activity, disability, and health-related quality of life (HRQOL) differences between children with juvenile idiopathic arthritis (JIA) and their healthy peers, and between children with JIA with and without clinical temporomandibular joint (TMJ) involvement and its determinants. METHODS: This study is based on a cross-sectional cohort of 3,343 children with JIA and 3,409 healthy peers, enrolled in the Pediatric Rheumatology International Trials Organisation HRQOL study or in the methotrexate trial. Potential determinants of TMJ involvement included demographic, disease activity, disability, and HRQOL measures selected through univariate and multivariable logistic regression. RESULTS: Clinical TMJ involvement was observed in 387 of 3,343 children with JIA (11.6%). Children with TMJ involvement, compared to those without, more often had polyarticular disease course (95% versus 70%), higher Juvenile Arthritis Disease Activity Score (odds ratio [OR] 4.6), more disability, and lower HRQOL. Children with TMJ involvement experienced clearly more disability and lower HRQOL compared to their healthy peers. The multivariable analysis showed that cervical spine involvement (OR 4.6), disease duration >4.4 years (OR 2.8), and having more disability (Childhood Health Assessment Questionnaire Disability Index >0.625) (OR 1.6) were the most important determinants for TMJ involvement. CONCLUSION: Clinical TMJ involvement in JIA is associated with higher disease activity, higher disability, and impaired HRQOL. Our findings indicate the need for dedicated clinical and imaging evaluation of TMJ arthritis, especially in children with cervical spine involvement, polyarticular course, and longer disease duration.

Hypermobility disorders in children and adolescents.

It is well recognized that many if not most children and adolescents attending paediatric rheumatology clinics will have a non-inflammatory origin for their complaints or disorder. Mechanical causes are frequently identified, and hypermobility or ligamentous laxity of joints is increasingly recognized as an aetiological factor in the presentation. Such conditions include 'growing pains', recurrent lower-limb arthralgia, anterior knee pain syndromes, and back pain. Studies of significant cohorts of such patients have now been published supporting the link of ligamentous laxity to particular symptom complexes. However, much disagreement remains as to the validity of hypermobility as an aetiogical factor. What seems clear is that not all hypermobile individuals will be symptomatic or indeed possibly have any risk for specific musculoskeletal disorders in later life. Screening tools such as the Beighton score are likely to be inadequate in many paediatric populations. Along with increasing recognition of these disorders in childhood and adolescence has been the development of a multidisciplinary management approach, which usually involves predominantly allied health professionals such as podiatrists, physiotherapists and occupational therapists. The challenge remains to interpret symptoms correctly as being related to the hypermobility and to predict why such children become symptomatic. The answer is likely to involve physiological and psychosocial factors. In addition, early identification and modification of risk factors may have major implications for subsequent prevalence of many adult medical disorders such as low back pain, chronic pain syndromes and degenerative osteoarthritis.

Management of Children with Juvenile Idiopathic Arthritis.

Juvenile idiopathic arthritis (JIA) comprises a group of heterogeneous disorders of chronic arthritis in childhood and remains the commonest pediatric rheumatic disease associated with significant long-term morbidity. Advances in understanding of the pathogenesis, better definition of disease control/remission measures, and the arrival of biological agents have improved the outcomes remarkably. Methotrexate (Mtx) remains the first-line disease modifying (DMARD) therapy for most children with JIA due to its proven efficacy and safety. Sulphosalazine (SSz) (especially for enthesitis) and leflunomide may also have a secondary role. Tumor necrosis factor inhibitors (TNF-I), alone or in combination with Mtx have shown tremendous benefit in children with polyarticular JIA, enthesitis related arthritis (ERA) and psoriatic arthritis. Tocilizumab appears very efficacious in systemic arthritis and abatacept and tocilizumab also appear to benefit polyarticular JIA; the role of rituximab remains unclear, though clearly beneficial in adult RA. TNF-I with Mtx is also effective in uveitis associated with JIA. Biologicals have demonstrated an impressive safety record in children with JIA, although close monitoring for rare but potentially dangerous adverse events, such as tuberculosis and other infections; paradoxical development of additional autoimmune diseases; and possibly an increased risk of cancers is warranted.

Analysis of 1p, 19q, 9p, and 10q as prognostic markers for high-grade astrocytomas using fluorescence in situ hybridization on tissue microarrays from Radiation Therapy Oncology Group trials.

Survival periods vary considerably for patients with high-grade astrocytomas, and reliable prognostic markers are not currently available. We therefore investigated whether genetic losses from chromosomes 1p, 19q, 9p, or 10q were associated with survival in 89 high-grade astrocytomas using tissue microarrays (TMAs) derived from Radiation Therapy Oncology Group clinical trials. Cases included 15 anaplastic astrocytomas (AAs) and 74 glioblastomas (GBMs) selected on the basis of survival times significantly shorter or longer than the expected median. Genetic analysis was performed by TMA-fluorescence in situ hybridization (FISH) on array sections using 8 DNA probes, including those directed at 1p32, 19q13.4, 9p21 (p16/CDKN2A), and 10q (PTEN and DMBT1). Genetic status for each locus was correlated with patient survival group, and data were analyzed by using Fisher's exact test of association (adjusted P = 0.025). Losses of chromosome 1p, either alone or in combination with 19q, were encountered in only 2 cases, both AAs. This contrasts with oligodendrogliomas, in which combined 1p and 19q losses are frequent and predictive of prolonged survival. Solitary 19q loss was noted in 3/15 AAs and in 7/70 GBMs and was more frequent in the long-term survival group (P = 0.041, AA and GBM combined). Chromosome 9p loss was seen in 5/8 AAs and 39/57 GBMs, whereas chromosome 10q loss was detected in 4/15 AAs and 48/68 GBMs. The 9p and 10q deletions were slightly more frequent in short-term survivors, though none of the comparisons achieved statistical significance. Long-term and short-term survival groups of high-grade astrocytomas appear to have dissimilar frequencies of 19q, 9p, and 10q deletions. TMA-FISH is a rapid and efficient way of evaluating genetic alterations in such tumors.

Scleroderma in children and adolescents.

The scleroderma disorders remain challenging conditions in pediatric rheumatology to understand pathologically and indeed for which to provide care. It is clear that much progress is being made in the clinical approach to understanding this group of group of disorders. It seems likely that the different lesions of LS may represent unique immunopathogenic mechanisms or perhaps reflect unique genetic or other characteristics of the patients themselves. To take advantage of the revolution in therapies now occurring in rheumatology it is critical that controlled clinical trials are developed with appropriate agreed upon outcome measures for both localized and systemic disease.