Bone Remodeling Markers in Children with Acute Lymphoblastic Leukemia after Intensive Chemotherapy: The Screenshot of a Biochemical Signature.

PURPOSE: to investigate the effects of intensive chemotherapy and glucocorticoid (GC) treatment on bone remodeling markers in children with acute lymphoblastic leukemia (ALL). METHODS: A cross-sectional study was carried out in 39 ALL children (aged 7.64 ± 4.47) and 49 controls (aged 8.7 ± 4.7 years). Osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), osteocalcin (OC), C-terminal telopeptide of type I collagen (CTX), bone alkaline phosphatase (bALP), tartrate-resistant acid phosphatase 5b (TRACP5b), procollagen type I N-terminal propeptide (P1NP), Dickkopf-1 (DKK-1), and sclerostin were assessed. Statistical analysis was conducted using the principal component analysis (PCA) to study patterns of associations in bone markers. RESULTS: ALL patients showed significantly higher OPG, RANKL, OC, CTX, and TRACP5b than the controls (p ≤ 0.02). Considering ALL group, we found a strong positive correlation among OC, TRACP5b, P1NP, CTX, and PTH (r = 0.43-0.69; p < 0.001); between CTX and P1NP (r = 0.5; p = 0.001); and between P1NP and TRAcP (r = 0.63; p < 0.001). The PCA revealed OC, CTX, and P1NP as the main markers explaining the variability of the ALL cohort. CONCLUSIONS: Children with ALL showed a signature of bone resorption. The assessment of bone biomarkers could help identify ALL individuals who are most at risk of developing bone damage and who need preventive interventions.

MUC1 Expression Affects the Immunoflogosis in Renal Cell Carcinoma Microenvironment through Complement System Activation and Immune Infiltrate Modulation.

Mucin1 (MUC1), a glycoprotein associated with an aggressive cancer phenotype and chemoresistance, is aberrantly overexpressed in a subset of clear cell renal cell carcinoma (ccRCC). Recent studies suggest that MUC1 plays a role in modulating cancer cell metabolism, but its role in regulating immunoflogosis in the tumor microenvironment remains poorly understood. In a previous study, we showed that pentraxin-3 (PTX3) can affect the immunoflogosis in the ccRCC microenvironment by activating the classical pathway of the complement system (C1q) and releasing proangiogenic factors (C3a, C5a). In this scenario, we evaluated the PTX3 expression and analyzed the potential role of complement system activation on tumor site and immune microenvironment modulation, stratifying samples in tumors with high (MUC1H) versus tumors with low MUC1 expression (MUC1L). We found that PTX3 tissue expression was significantly higher in MUC1H ccRCC. In addition, C1q deposition and the expressions of CD59, C3aR, and C5aR were extensively present in MUC1H ccRCC tissue samples and colocalized with PTX3. Finally, MUC1 expression was associated with an increased number of infiltrating mast cells, M2-macrophage, and IDO1+ cells, and a reduced number of CD8+ T cells. Taken together, our results suggest that expression of MUC1 can modulate the immunoflogosis in the ccRCC microenvironment by activating the classical pathway of the complement system and regulating the immune infiltrate, promoting an immune-silent microenvironment.

Determination of iFGF23 Upper Reference Limits (URL) in healthy pediatric population, for its better correct use.

Background: The measurement of Fibroblast growth factor 23 (FGF23) may be useful in the diagnosis and management of abnormal phosphate metabolism in both patients with preserved renal function or with chronic kidney disease (CKD). FGF-23 tests differ considerably by molecule assayed (iFGF23 or cFGF23), analytical performance and reference ranges. We establish iFGF23 Upper Reference Limits (URL) in apparently healthy pediatric individuals using automated immunochemiluminescent assay. Methods: We measured the levels of plasma iFGF23 from 115 samples from apparently healthy pediatric subjects [59 (51.3%) individuals were male; median age 10 years (range 1-18)] included in an observational study conducted at Policlinico University Hospital of Bari. The method used for the iFGF23 assay was immunochemiluminescent sandwich assay developed by DiaSorin on the Liaison XL platform. Statistical calculation of 95% reference interval, right-sided (CLSI C28-A3) and verification of age and sex covariables was performed for the calculation of the URL. Results: The URL concentration of iFGF23 was 61.21 pg/mL (58.63 to 63.71, 90% CI). No significant differences were found between the median concentrations of iFGF23 differentiated by sex and age. Conclusions: The dosage of iFGF23 is important both for the differential diagnosis of the various forms of rickets, and for the subsequent monitoring of the effectiveness of drug treatment. We have established the URL for the iFGF23 Liaison test in apparently healthy pediatric subjects. The availability of iFGF23 pediatric reference values will allow a better clinical use of the test.

Parathyroid Hormone-Related Peptide (PTHrP): Evaluation of Pediatric, Covariate-Stratified Reference Intervals.

Parathyroid hormone-related peptide (PTHrP) is expressed at a wide range of sites in the body and performs different functions including vasodilation, relaxation of smooth muscle cells, and regulation of bone development. PTHrP also mediates hypercalcemia related to neoplastic diseases. However, reference ranges specific method and age were not evaluated. We establish PTHrP reference ranges in apparently healthy, normocalcemic, normophosphatemic pediatric individuals. In this observational prospective, study we measured PTHrP in serum from 178 samples (55.06% male 44.94% female) from apparently healthy pediatric subjects [median age 10 years (range 1-18)] subunit ELISA method The statistical analysis performed provided for the calculation of the 95% reference interval, right-sided, with a non-parametric percentile method (CLSI C28-A3). Upper reference limits (URL) for PTHrP was 2.89 ng/mL (2.60 to 3.18; 90% CI). No significant differences were found between the median PTHrP concentrations in males vs females and in the age range categories selected. Comprehensive normal values for PTHrP are indispensable to the assessment of calcium phosphorus dysfunction in children. Severe hypercalcemia is a rare, but clinically significant condition, in infancy and childhood. PTHrP values higher than the reference value may help to distinguish the hypercalcemic product of a malignancy, paraneoplastic syndromes mediated by PTHrP, from other causes.

Irisin Serum Levels and Skeletal Muscle Assessment in a Cohort of Charcot-Marie-Tooth Patients.

Background: Charcot-Marie-Tooth (CMT) indicates a group of inherited polyneuropathies whose clinical phenotypes primarily include progressive distal weakness and muscle atrophy. Compelling evidence showed that the exercise-mimetic myokine irisin protects against muscle wasting in an autocrine manner, thus possibly preventing the onset of musculoskeletal atrophy. Therefore, we sought to determine if irisin serum levels correlate with biochemical and muscle parameters in a cohort of CMT patients. Methods: This cohort study included individuals (N=20) diagnosed with CMT disease. Irisin and biochemical markers were quantified in sera. Skeletal muscle mass (SMM) was evaluated by bioelectric impedance analysis, muscle strength by handgrip, and muscle quality was derived from muscle strength and muscle mass ratio. Results: CMT patients (m/f, 12/8) had lower irisin levels than age and sex matched healthy subjects (N=20) (6.51 ± 2.26 vs 9.34 ± 3.23 µg/ml; p=0.003). SMM in CMT patients was always lower compared to SMM reference values reported in healthy Caucasian population matched for age and sex. Almost the totality of CMT patients (19/20) showed low muscle quality and therefore patients were evaluated on the basis of muscle strength. Irisin was lower in presence of pathological compared to normal muscle strength (5.56 ± 1.26 vs 7.67 ± 2.72 µg/ml; p=0.03), and directly correlated with the marker of bone formation P1PN (r= 0.669; 95%CI 0.295 to 0.865; p=0.002), but inversely correlated with Vitamin D (r=-0.526; 95%CI -0,791 to -0,095; p=0.017). Surprisingly, in women, irisin levels were higher than in men (7.31 ± 2.53 vs 5.31 ± 1.02 µg/ml, p=0.05), and correlated with both muscle strength (r=0.759; 95%CI 0.329 to 0.929; p=0.004) and muscle quality (r=0.797; 95%CI 0.337 to 0.950; p=0.006). Conclusion: Our data demonstrate lower irisin levels in CMT patients compared to healthy subjects. Moreover, among patients, we observed, significantly higher irisin levels in women than in men, despite the higher SMM in the latter. Future studies are necessary to establish whether, in this clinical contest, irisin could represent a marker of the loss of muscle mass and strength and/or bone loss.

Evaluation of Vitamin D (25OHD), Bone Alkaline Phosphatase (BALP), Serum Calcium, Serum Phosphorus, Ionized Calcium in Patients with Mandibular Third Molar Impaction. An Observational Study.

The aim of this study was to evaluate the levels of vitamin D (25OHD) and other bone biomarkers in patients with third molar impaction (TMI). Thirty males and 30 females with unilateral or bilateral impacted mandibular third molar, and 15 males and 15 females as a control group (CG) were recruited. Rx-OPT was used to evaluate dental position and Pederson index to measure the difficulty of the intervention. Bone biomarkers were measured through blood venous sample in TMI group and CG. Mann-Whitney test, Pearson's correlation coefficient, linear regression model were used to compare the different parameters in the two groups. 25OHD showed lower values in TMI group than in CG (p < 0.05) with values significantly lower in bilateral impaction (p < 0.05). Pearson's coefficient for 25OHD presented a negative correlation with the Pederson index (ρ = -0.75). Bone alkaline phosphatase (BALP) showed significantly lower dosage in TMI group than CG (p = 0.02), Pearson's coefficient for BALP presented a negative correlation with the Pederson index. Serum calcium, serum phosphorus, ionized calcium levels in TMI and CG groups were similar and Mann-Whitney test did not significantly differ between TMI and CG. TMI could be a sign of vitamin D deficiency and of low BALP levels that should be investigated.

A New Immunodot Assay for Multiplex Detection of Autoantibodies in a Cohort of Italian Patients With Idiopathic Inflammatory Myopathies.

BACKGROUND: Autoantibody detection has been assessed as tool for the diagnosis and the definition of idiopathic inflammatory myopathies (IIM). The aim of the study was to characterize the autoantibody profiling of a cohort of Italian patients with IIM. METHODS: Sera of 53 adult patients with definite IIM, according to Bohan-Peter criteria, were tested for anti-nuclear autoantibodies (ANA), using indirect immunofluorescence (IIF) method, and for myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs), using two new commercial immunodot assays. RESULTS: MSAs and/or MAAs were detected in 29 of 53 (54.7%) patients with IIM. Twenty-three patients (43.4%) were positive for at least one MSAs: 13 (24.5%) had anti-histidyl-tRNA synthetase autoantibodies (Jo1), 4 (7.5%) had other anti-aminoacyl-tRNA synthetases autoantibodies (anti-ARS), 1 (1.8%) had anti-transcription intermediary factor 1 gamma autoantibodies (anti-TIF1γ), 2 (3.7%) had anti-nuclear helicase protein Mi-2 autoantibodies (anti-Mi-2), 4 (7.5%) had anti-small ubiquitin like modifier activating enzyme heterodimer autoantibodies (anti-SAE). Moreover, 17 patients (32%) were positive for at least one MAAs. Coexisting MSAs and MAAs were observed in 9 of 53 (16.9%) patients, anti-Jo1/SS-A autoantibodies in most cases. Overall sensitivity of immunodot assays was 54.7%, the specificity was almost absolute. At cut-off value of 1:160, the sensitivity of ANA-IIF was 52.8%, increasing to 66% if cytoplasmatic fluorescence reaction was reported. Notably, two (5.7%) ANA-IIF negative patients had MSAs, detected only by immunodot assays. CONCLUSION: It was possible to identify MSAs otherwise undetectable because of the use of new assays. Immunodot can reveal MSAs even when IIF results are inconclusive or, in some cases, ANA negative.

Usefulness of tissue transglutaminase type 2 antibodies in early pregnancy.

Celiac disease (CD), an autoimmune disease triggered by dietary gluten, is a multi-systemic disorder that primarily results in mucosal damage of the small intestine. Reproductive disorders and pregnancy complications have been associated with CD. Conflicting results have been published concerning CD and the risk of impaired fetal growth with reduced birthweight. The aim of our multicentric, perspective, case-control study was to determine the prevalence of undiagnosed CD in mothers of small for gestational age (SGA) newborns in two regions of Italy. The study included 480 mothers: group A consisted of 284 SGA newborns' mothers and group B consisted of 196 appropriate for gestational age (AGA) newborns' mothers. Tissue transglutaminase type 2 antibodies (TG2) IgA and IgG were measured in blood samples. We diagnosed two new cases of CD in asymptomatic mothers. It may be appropriate to include the TG2 to the panel of prenatal blood test.

Biomarkers in malignant mesothelioma: diagnostic and prognostic role of soluble mesothelin-related peptide.

Soluble mesothelin-related peptide (SMRP) is a biomarker that has been proposed for differential diagnosis from pleural metastatic cancer, as well as prognosis and treatment monitoring of malignant pleural mesothelioma (MM). The aim of this study was to evaluate the role of SMRP in clinic management of MM. We assayed the SMRP concentrations in 354 subjects: 109 healthy volunteers with no history of exposure to asbestos, 26 patients with previous occupational asbestos exposure but who were free from pleural or parenchymal disease, 48 patients with asbestosis, 110 patients with pleural plaques, 25 patients with lung cancer, and 36 patients with MM. We also tested SMRP titers in 2 patients with MM at 5 different times of the disease, to evaluate the trend of the biomarker in the course of therapy. Our data confirm previous experiences with the use of SMRP as a diagnostic marker of MM. Low SMRP levels at diagnosis seem to have a positive prognostic significance.

Evaluation of a new ELISA assay for detection of BP230 autoantibodies in bullous pemphigoid.

The diagnosis of bullous pemphigoid is based on clinical observations and on the presence of autoantibodies directed against proteins of the dermoepidermal junction. Human recombinant BP180 and BP230 peptides have been used to develop new quantitative enzyme immunoassays (EIA) for the detection of specific antibodies. This study evaluated the sensitivity and specificity of a new immunoassay for the detection of BP230 autoantibodies and clinical correlations. Serum samples were tested from patients with bullous pemphigoid, other skin diseases, and from healthy donors. Autoantibodies anti-BP230 and anti-BP180 were assayed using the EIA method. Diagnostic specificity for both tests was over 98%; diagnostic sensitivity was 90% and 60% for anti-BP180 and anti-BP230, respectively. IgG anti-BP180 titers exhibited a significant correlation with disease activity. No patient in remission was positive for anti-BP230. In conclusion, anti-BP180 and anti-BP230 assays are useful in the diagnosis of bullous pemphigoid and provide information on disease activity.

Mesothelin family proteins and diagnosis of mesothelioma: analytical evaluation of an automated immunoassay and preliminary clinical results.

BACKGROUND: Studies have suggested that soluble mesothelin-related protein (SMRP) can be used as a serum marker of malignant mesothelioma. METHODS: We assessed the analytical performance of the Mesomark (Fujirebio Diagnostic) two-step ELISA on an automated analyser and performed a preliminary clinical evaluation. The precision of the assay and the in vitro effect of interfering substances on SMRP concentrations were investigated. The serum marker was analysed in 109 healthy subjects never exposed to asbestos, 26 healthy subjects exposed to asbestos, 33 patients with asbestosis, 33 with asbestos-related pleural plaques, 10 with non-malignant pleural diseases, 30 with lung cancer and 24 with histological diagnosis of pleural mesothelioma. RESULTS: Using the International Mesothelioma Interest Group classification, there were nine stage IV, two stage III, four stage II and nine stage I patients. SMRP concentrations of 4.75, 11.0 and 14 nmol/mL showed a total imprecision of 3.5%, 3.1% and 3.8%. The detection limit was 0.035 nmol/mL; the mean SMRP concentration of 0.63 nmol/mL was associated with a coefficient of variation of 10%. There was no effect (p>0.05) of interfering substances. Serum samples from patients with established pleural mesothelioma had significantly higher (p<0.05) concentrations of SMRP than control healthy and patient groups. CONCLUSIONS: SMRP measured on automated systems could be useful for the diagnosis of mesothelioma in routine clinical practice.

Application of a combined protocol for rational request and utilization of antibody assays improves clinical diagnostic efficacy in autoimmune rheumatic disease.

CONTEXT: Because of a marked increase in the number of requests for antinuclear antibodies, anti-extractable nuclear antigen antibodies, and anti-double-stranded DNA antibodies for the diagnosis of autoimmune rheumatic disease, guidelines have been proposed for their appropriate use. OBJECTIVE: To evaluate in terms of clinical efficacy and cost-benefit ratio the outcome of applying a protocol for the diagnosis of autoimmune rheumatic disease. DESIGN: A diagnostic protocol for the rational utilization of second-level tests (anti-extractable nuclear antigen antibodies and anti-double-stranded DNA antibodies) was applied at Hospital Polyclinic beginning January 2004. The appropriateness of 685 consecutive requests received at the clinical pathology laboratory from January to June 2004 was assessed. Patients who underwent these laboratory tests were followed up for 12 months after blood sample drawing. RESULTS: Introduction of the protocol led to a significant reduction in the number of second-level tests prescribed (27.9% vs 49.5% for anti-extractable nuclear antigen antibodies; 27.5% vs 56.6% for anti-double-stranded DNA antibodies). After the period of observation, none of the 163 patients who had negative results on the first-level test and were asymptomatic, for whom second-level tests had not therefore been performed, were found to have autoimmune rheumatic disease. In 90.5% (77/85) of patients positive for the second-level tests, clinical confirmation of autoimmune rheumatic disease was obtained. CONCLUSIONS: Not only did application of the diagnostic protocol reduce the number of second-level tests performed but it also increased their specificity. Our data thus indicate that the use of shared guidelines by clinical and laboratory specialists yields satisfactory results.

Anti-cyclic citrullinated peptide autoantibodies measured by an automated enzyme immunoassay: analytical performance and clinical correlations.

BACKGROUND: Autoantibodies against cyclic citrullinated peptide (anti-CCP) are considered to be a sensitive and specific marker for rheumatoid arthritis (RA). This study evaluated the analytical performance and clinical correlation of an automated enzyme immunoassay (DSX, DINEX Technologies), for the detection of anti-CCP autoantibodies (DIASTAT anti-CCP, Axis-Shield, DUNDEE UK). METHODS: Commercial controls and serum pools were used to determine its precision, analytical sensitivity, functional sensitivity and linearity. Sera from 83 patients with established RA and from 140 controls, including patients with various autoimmune diseases, viral infections and cancer, as well as sex- and age-matched healthy subjects, were studied. The rheumatoid factor (RF) was also assayed in each sample, and the results were compared to the anti-CCP findings. RESULTS: The total imprecision (CV%) was 4.7-7.2% for concentrations ranging between 1.98 and 71.81 U/mL. The lower detection limit was 0.038 U/mL. At a cut-off of 5 U/mL, the sensitivity and specificity for RA were 67.5% and 99.3%, respectively. The RF had a sensitivity of 66.3% and a lower specificity 82.1% than anti-CCP. When the two antibodies were used together, the specificity was 99.1%. CONCLUSIONS: The anti-CCP assay we examined on a fully automated system showed a good analytical performance (analytical and functional sensitivity, linearity) and good clinical correlation. We conclude that this system can provide rapid, useful data.