Pancreatic cancer biomarker detection by two support vector strategies for recursive feature elimination.

AIM: Pancreatic cancer is one of the worst malignant tumors in prognosis. Therefore, to reduce the mortality rate of pancreatic cancer, early diagnosis and prompt treatment are particularly important. RESULTS: We put forward a new feature-selection method that was used to find clinical markers for pancreatic cancer by combination of Support Vector Machine Recursive Feature Elimination (SVM-RFE) and Large Margin Distribution Machine Recursive Feature Elimination (LDM-RFE) algorithms. As a result, seven differentially expressed genes were predicted as specific biomarkers for pancreatic cancer because of their highest accuracy of classification on cancer and normal samples. CONCLUSION: Three (MMP7, FOS and A2M) out of the seven predicted gene markers were found to encode proteins secreted into urine, providing potential diagnostic evidences for pancreatic cancer.

Discovery and Qualification of Candidate Urinary Biomarkers of Disease Activity in Lupus Nephritis.

Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Noninvasive biomarkers are needed to inform treatment decisions and to monitor disease activity. Proteinuria is associated with disease progression in LN; however, the composition of the LN urinary proteome remains incompletely characterized. To address this, we profiled LN urine samples using complementary mass spectrometry-based methods:  protein gel fractionation, chemical labeling using tandem mass tags, and data-independent acquisition. Combining results from these approaches yielded quantitative information on 2573 unique proteins in urine from LN patients. A multiple-reaction monitoring (MRM) method was established to confirm eight proteins in an independent cohort of LN patients, and seven proteins (transferrin, α-2-macroglobulin, haptoglobin, afamin, α-1-antitrypsin, vimentin, and ceruloplasmin) were confirmed to be elevated in LN urine compared to healthy controls. In this study, we demonstrate that deep mass spectrometry profiling of a small number of patient samples can identify high-quality biomarkers that replicate in an independent LN disease cohort. These biomarkers are being used to inform clinical biomarker strategies to support longitudinal and interventional studies focused on evaluating disease progression and treatment efficacy of novel LN therapeutics.

Urine protein biomarkers for the diagnosis and prognosis of necrotizing enterocolitis in infants.

OBJECTIVES: To test the hypothesis that an exploratory proteomics analysis of urine proteins with subsequent development of validated urine biomarker panels would produce molecular classifiers for both the diagnosis and prognosis of infants with necrotizing enterocolitis (NEC). STUDY DESIGN: Urine samples were collected from 119 premature infants (85 NEC, 17 sepsis, 17 control) at the time of initial clinical concern for disease. The urine from 59 infants was used for candidate biomarker discovery by liquid chromatography/mass spectrometry. The remaining 60 samples were subject to enzyme-linked immunosorbent assay for quantitative biomarker validation. RESULTS: A panel of 7 biomarkers (alpha-2-macroglobulin-like protein 1, cluster of differentiation protein 14, cystatin 3, fibrinogen alpha chain, pigment epithelium-derived factor, retinol binding protein 4, and vasolin) was identified by liquid chromatography/mass spectrometry and subsequently validated by enzyme-linked immunosorbent assay. These proteins were consistently found to be either up- or down-regulated depending on the presence, absence, or severity of disease. Biomarker panel validation resulted in a receiver-operator characteristic area under the curve of 98.2% for NEC vs sepsis and an area under the curve of 98.4% for medical NEC vs surgical NEC. CONCLUSIONS: We identified 7 urine proteins capable of providing highly accurate diagnostic and prognostic information for infants with suspected NEC. This work represents a novel approach to improving the efficiency with which we diagnose early NEC and identify those at risk for developing severe, or surgical, disease.

Urinary IgG and α2-macroglobulin are powerful predictors of outcome and responsiveness to steroids and cyclophosphamide in idiopathic focal segmental glomerulosclerosis with nephrotic syndrome.

OBJECTIVE: To assess whether high-molecular-weight proteins excretion predicts outcome and therapy-responsiveness in patients with FSGS and nephrotic syndrome. RESEARCH DESIGN AND METHODS: Thirty-eight patients measured at biopsy fractional excretion of IgG (FEIgG) and urinary α2-macroglobulin/creatinine ratio ( α m/C). Low and high risk groups were defined by cutoffs assessed by ROC analysis. In all patients first-line therapy was with steroids alone or in combination with cyclophosphamide. RESULTS: α2m/C and FEIgG were correlated with segmental sclerosis (r = 0.546; r = 0.522). Twenty-three patients (61%) entered Remission and 9 (24%) progressed to ESRD. Comparing low and high risk groups, by univariate analysis remission was predicted by FEIgG (77% versus 25%, P = 0.016) and α2m/C (81% versus 17%, P = 0.007) and ESRD at best by FEIgG (0% versus 75%, P < 0.0001) and α2m/C (4% versus 67%, P < 0.0001). By multivariate analysis FEIgG was the only independent predictor of remission and α2m/C the most powerful predictor of ESRD. Low and high risk groups of FEIgG and α2m/C in combination had very high predictive value of sustained remission and ESRD in response to therapy. CONCLUSIONS: FEIgG and α2m/C are powerful predictors of outcome and responsiveness to steroids and cyclophosphamide; their predictive value, if validated in prospective studies, may be useful in clinical practice suggesting first-line alternative treatments in high risk patients.

[Significance of urinary biomarkers in differential diagnosis of acute tubulointerstitial nephritis].

OBJECTIVE: To find some urinary biomarkers with significance in the differentiation of drug-induced tubulointerstitial nephritis (DTIN). METHODS: Forty patients with biopsy-proven DTIN were enrolled. The urine samples of DTIN patients were collected on the day of biopsy and all urine samples were measured for the following different biomarkers as indicated, respectively: urinary TGF-beta by ELISA; urinary IL-6 by radio-immunoassay; NAG by an enzyme-substrate colorimetric assay; alpha1-MG by immune transmission turbidity method. Receiver operating characteristic curves (ROC curve) were constructed to calculate the sensitivity and specificity of those biomarkers in distinguishing acute (A-DTIN) and chronic DTIN (C-DTIN). RESULTS: Urinary NAG and alpha1-MG levels in patients with A-DTIN were as 2.5 and 2.1 times as those in C-DTIN (P<0.05), while urinary TGF-beta levels in the two groups had no statistical difference. The areas under ROC curve (AUC) of urinary NAG and alpha1-MG for differentiating A-DTIN were 0.720 (P=0.029) and 0.714 (P=0.034) respectively, while the AUCs for TGF-beta and IL-6 were 0.536 (P=0.767) and 0.150 (P=0.004) respectively. Combined measurement of NAG and alpha1-MG could make sensitivity and specificity reach 78.6% and 75.0 % respectively. CONCLUSION: Urinary alpha1-MG and NAG levels can reflect the acute lesions of DTIN, and combined measurement of both could enhance efficiency in differentiating A-DTIN.

Comparative analysis of urinary biomarkers for early detection of acute kidney injury following cardiopulmonary bypass.

The purpose of this study was to compare the performance of six candidate urinary biomarkers, kidney injury molecule (KIM)-1, N-acetyl-beta-D-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL), interleukin (IL)-18, cystatin C and alpha-1 microglobulin, measured 2 h following cardiopulmonary bypass (CPB) for the early detection of acute kidney injury (AKI) in a prospective cohort of patients undergoing cardiac surgery. A total of 103 subjects were enrolled; AKI developed in 13%. Urinary KIM-1 achieved the highest area under-the-receiver-operator-characteristic curve (AUC 0.78, 95% confidence interval 0.64-0.91), followed by IL-18 and NAG. Only urinary KIM-1 remained independently associated with AKI after adjustment for a preoperative AKI prediction score (Cleveland Clinic Foundation score; p = 0.02), or CPB perfusion time (p = 0.006). In this small pilot cohort, KIM-1 performed best as an early biomarker for AKI. Larger studies are needed to explore further the role of biomarkers for early detection of AKI following cardiac surgery.

In crescentic IgA nephropathy, fractional excretion of IgG in combination with nephron loss is the best predictor of progression and responsiveness to immunosuppression.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the relationship between proteinuric markers (urinary excretion of IgG, alpha2-macroglobulin, alpha1-microglobulin) and serum creatinine (sCr), histologic lesions, progression, and immunosuppression responsiveness in crescentic IgA nephropathy. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Fractional excretion of IgG (FEIgG) and of alpha1-microglobulin and urinary excretion of alpha2-macroglobulin were evaluated in 37 patients, 23 treated with steroids and cyclophosphamide. For assessment of the effective tubular load of proteins in surviving nephrons, new markers that take into account not only the absolute excretion value but also nephron loss were obtained dividing proteinuric markers for percentage of nonobsolescent glomeruli (surviving glomeruli [SG]). For each parameter, low- and high-risk groups were defined according to cutoffs with the highest sensitivity and specificity for progression (ESRD/doubling sCr) assessed by receiver operating characteristic analysis; follow up was 60 +/- 40 mo. RESULTS: FEIgG/SG is the most powerful progression predictor: 5 versus 83% in all patients; in treated patients, 0 versus 89%, increased to 0 versus 100% by sCr and FEIgG/SG in combination (low risk: both markers or only one below cutoff (n = 15); high risk: both markers above cutoff (n = 8). The nonprogressors showed at last observation 65% proteinuria reduction and 10% sCr reduction. CONCLUSIONS: In crescentic IgA nephropathy, FEIgG/SG, which evaluates altered size selectivity in relation to nephron loss, is the best progression predictor. In treated patients, progression prediction was increased by FEIgG/SG and sCr in combination. Treatment may be restricted to low-risk patients.

[Evaluation of inflammatory and renal injury markers in youngest children with pyelonephritis]. / Ocena wskazników zapalenia i uszkodzenia nerek u najmlodszych dzieci chorych na odmiedniczkowe zapalenie nerek.

UNLABELLED: Pyelonephritis (PN) is frequent bacterial infections in young infants and very important because may cause parenchymal scarring. Early confirmation of bacterial infection and application the appropriate treatment before obtaining result of urine culture, reduce probability of parenchymal scarring. THE AIM OF THE STUDY: To evaluate the useful of inflammatory and renal injury markers: serum procalcitonin (PCT), tumor necrosis factor alpha (TNF-alpha) and injury renal marker alpha1--microglobulin (A1M) measurement, in comparison with C-reactive protein concentration and abnormal urinary tract, in neonates and young infants with pyelonephritis. MATERIAL AND METHODS: Investigation was performed in two groups: I group--23 children with PN (1 to 24 weeks of age), and K group--30 healthy children aged from 1 to 24 weeks. Serum concentration of CRP was measured by immunonephelometric assay, PCT by immunoluminometric assay, TNF alpha by ELISA method, and urinary A1M by nephelometric assay. RESULTS: In control group (K) medians of all investigated markers were below minimum of detection. PN patients (I) had the highest PCT TNF-alpha, A1M and CRP concentration before treatment and normal results after antibiotic treatment. Using a cut-off: of 0.5 mg/dl for CRP, 0.5 ng/ml for PCT 15 pg/ml for TNF-alpha and 10 mg/g cr for A1M, sensitivity and specificity in children with pyelonephritis were: for CRP 100% and 62.5%, for PCT 81.8% and 87.2%, for TNF alpha 77.1% and 93.1% and A1M 70.4% and 56.1%, respectively. A positive correlation between serum PCT and CRP and TNF alpha was found. Very high concentration all markers were in patients with vesicoureteral reflux and 1 patient with hydronephrosis. CONCLUSION: In early diagnostics of PN (before obtaining results of urine culture) in youngest children, determination of concentration PCT and TNF alpha, has higher value than determination of CRP, taking into concentration high sensitivity and specificity for bacterial infection.

Detection of subclinical tubular injury after renal transplantation: comparison of urine protein analysis with allograft histopathology.

BACKGROUND: Tubulointerstitial injury due to rejection leads to tubular atrophy (TA)/interstitial fibrosis (IF) followed by deterioration of allograft function. This study investigated whether urinary tubular injury biomarkers can detect subclinical tubulitis found in protocol biopsies allowing for a noninvasive screening procedure. METHODS: Four rigidly defined groups (stable transplants with normal tubular histology [n=24], stable transplants with subclinical tubulitis [n=38], patients with clinical tubulitis Ia/Ib [n=18], and patients with other clinical tubular pathologies [n=20]) were compared for differences in urinary intact/cleaved beta2-microglobulin (i/cbeta2m), retinol-binding protein (RBP), neutrophil-gelatinase-associated lipocalin (NGAL), and alpha1-microglobulin (alpha1m). RESULTS: Tubular proteinuria was present in 38% (RBP) to 79% (alpha1m) of patients in the stable transplant with normal tubular histology group. The stable transplant with subclinical tubulitis group had slightly higher levels of i/cbeta2m (P=0.11), RBP (P=0.17), alpha1m (P=0.09), and NGAL (P=0.06) than the stable transplant with normal tubular histology group with a substantial overlap. The clinical tubulitis Ia/Ib and the other clinical tubular pathology groups had significantly higher levels of RBP, NGAL, and alpha1m than stable transplants with normal tubular histology or stable transplants with subclinical tubulitis (P<0.002). CONCLUSIONS: None of the investigated biomarkers allow for clear differentiation between stable transplants with normal tubular histology and stable transplants with subclinical tubulitis. Therefore, the protocol allograft biopsy currently remains the preferred tool to screen for subclinical tubulitis. Further longitudinal studies should determine whether tubular proteinuria in stable transplants with normal tubular histology indicates a clear risk for early development of TA/IF.

Urinary plasma protein patterns in acute prostatitis.

We evaluated the diagnostic utility of urinary alpha1-microglobulin, alpha2-macroglobulin and albumin in the diagnosis of acute prostatitis. We studied 133 men (43 +/- 17 years) with, and a reference population (n=36, 41 +/- 16 years) without, urinary tract infection. Prostatectomy samples were used to study the potential interference between prostatic proteins and protein analysis. Urinary alpha2-macroglobulin/albumin ratio was significantly lower in prostatitis compared to the reference population, cystitis or acute pyelonephritis (p < 0.0001). Low alpha2-macroglobulin concentrations in prostatitis are due to inhibition (p = 0.0001) of the immune reaction between alpha2-macroglobulin in presence of polyclonal rabbit antibodies (used for immunonephelometry) by soluble prostatic proteins (+/- 60 kDa) which appear in urine in acute prostatitis. The urinary alpha1-microglobulin/creatinine ratio diagnoses acute pyelonephritis (sensitivity 100% and specificity 87%) and the urinary alpha2-macroglobulin/albumin ratio diagnoses acute prostatitis (sensitivity 100% and specificity of 90%). Stepwise multinomial logistic regression analysis reveals that urinary alpha1-microglobulin, alpha2-macroglobulin, albumin and creatinine provide optimal differentiation between acute pyelonephritis and acute prostatitis (pseudo R2=0.83; Loglikelihood -30.55, p < 0.000001). In conclusion, the combination of hematuria and absence of urinary alpha-2-macroglobulin is diagnostic for acute prostatitis. Even without hematuria, alpha2-macroglobulin remains lower compared to patients without prostatitis.

Proteinuria selectivity index based upon alpha 2-macroglobulin or IgM is superior to the IgG based index in differentiating glomerular diseases. Technical note.

BACKGROUND: The proteinuria selectivity index (SI) may be used to describe changes of the glomerular permeability for macromolecules in glomerular diseases. Proteins the size of alpha 2-macroglobulin (alpha 2 M) or IgM cannot normally pass the glomerular barrier, whereas IgG can pass through the large pores of glomerular basement membrane. Comparison of the clearance of the three high-molecular-weight proteins to that of albumin may be useful in characterization and diagnosis of different glomerular diseases as well as in understanding of the permeability characteristics of the glomerular filter. METHODS: Three types of SI, each calculated as a ratio of clearance of either IgG, alpha 2M or IgM to that of albumin, were measured in 199 proteinuric patients. The patients were subdivided into eight different biopsy-verified glomerular diseases. RESULTS: Two diagnoses could be clearly distinguished using SI based on alpha 2M (alpha 2 M SI) or IgM (IgM SI). Both alpha 2M SI and IgM SI were significantly lower in minimal change nephropathy and higher in crescentic necrotizing glomerulonephritis than in all the other diagnoses. The SI based on IgG (IgG SI) was less useful in determining specific diagnoses, since patients with minimal change nephropathy could not be distinguished from those with other types of primary glomerulonephritis and patients with crescentic necrotizing glomerulonephritis did not differ from those with diabetic nephropathy. CONCLUSIONS: The findings of this study indicate that alpha 2M SI and IgM SI are superior to IgG SI in characterization of glomerular disorders and might replace the IgG SI for this purpose.

Differentiation of hematuria by quantitative determination of urinary marker proteins.

Hematuria caused by prerenal, glomerular, postglomerular, and postrenal causes is usually differentiated by a number of noninvasive and invasive diagnostic procedures. In the present study we have applied a new analytical strategy based on observations that the various forms of hematuria can be classified by their typical protein pattern. When analyzed by quantitative turbidimetric assays, urines from postrenal hematurias contained high-molecular-weight proteins (alpha 2-macroglobulin and IgG) in proportions found in plasma. Relating excretion rates (mg/mg) of these proteins to those of albumin, ratios for alpha 2-macroglobulin/albumin and IgG/albumin were 2.0-31 x 10(-2) and 20.0-180 x 10(-2), respectively. In contrast, glomerular hematurias exhibited ratios of 0.01-2.0 x 10(-2) (alpha 2-macroglobulin/albumin) and 2.0-20 x 10(-2) (IgG/albumin). Additional determination of alpha 1-microglobulin allowed us to differentiate postglomerular hematurias caused by interstitial nephropathies from glomerular and postrenal diseases. Critical evaluation of 93 cases diagnosed by independent clinical examination including histology, sonography, and cystoscopy revealed that the criteria derived from protein measurements resulted in correct classification when urine albumin exceeds 100 mg/l. This noninvasive procedure is expected to be of considerable help in the primary care of patients with unexplained hematuria.

Detection of lysozyme and alpha 2-macroglobulin--lysozyme complexes by immunoblotting.

An immunoblotting technique was developed to detect human lysozyme and lysozyme complexes in body fluids. The unoccupied binding capacity of proteins was demonstrated by addition of surplus lysozyme. The sensitivity of immunoblotting to the free enzyme in human albumin solution was less than 5 ng. In serum and pleural fluid, part of exogenous lysozyme was bound to alpha 2-macroglobulin (alpha 2-M). At high concentrations of lysozyme in leukemic sera, part of the enzyme formed an endogenous alpha 2-M complex. On the other hand, the formation of alpha 2-M complexes with exogenous lysozyme was especially striking in sera from nephrotic patients with elevated alpha 2-M. The findings corroborate with previous reports on lysozyme binding to purified alpha 2-M in vitro and suggest that the binding is concentration-dependent with respect to both reaction partners. In vivo the mechanism may provide a pathway for extrarenal lysozyme catabolism medicated by reticuloendothelial cells. No other binding proteins were seen in the present study: lysozyme did not bind to serum immunoglobulins in 35 samples with an immunoglobulin paraprotein, three samples with polyclonally elevated gamma-globulins, 20 other patient sera and 10 normal sera. Neither did lysozyme bind to urinary proteins in five samples from patients with myeloic leukemias nor in 10 samples from myeloma patients with urinary excretion of a monoclonal immunoglobulin light chain.

Epidemiology of renal tubular dysfunction in the inhabitants of a cadmium-polluted area in the Jinzu River basin in Toyama Prefecture.

Urinary beta 2-microglobulin (beta 2-m), alpha 1-microglobulin (alpha 1-m), amino-nitrogen, glucose, calcium, phosphorus, cadmium concentrations, and pH values were analyzed in urine samples from 187 females aged 55-66 years in the Jinzu River basin, which is known to be a cadmium-polluted area, and from 32 controls living in two adjacent reference areas in 1983-1984. Mean urinary beta 2-m, alpha 1-m, amino-nitrogen, glucose, cadmium concentrations and pH values in the inhabitants of the Jinzu River basin were significantly higher than those in the adjacent reference areas. Sixty-four inhabitants in a cadmium-polluted area were found to have renal tubular dysfunction with urinary beta 2-m level exceeding 1 mg/g creatinine and urinary glucose level exceeding 100 mg/g creatinine. The severity of renal tubular dysfunction in several inhabitants were comparable to those of the patients with Itai-itai disease. Mean cadmium concentrations in rice (mean: 0.32-0.57 ppm) which has been daily consumed by the inhabitants of the Jinzu River basin were significantly higher than those in the reference areas (mean: 0.12-0.13 ppm). The close relationship between cadmium exposure and the degree of renal tubular dysfunction was well demonstrated by principal component analysis.

Isoelectric points of urinary light chains in myelomatosis: analysis in relation to nephrotoxicity.

Measurements of the isoelectric point (pI), sialic acid content, and polymerisation were made of 43 light chains isolated from the urine of patients with myelomatosis excreting large amounts of light chains. The pIs ranged from 3.5-9.5, and 23 were greater than 7.0. Sialylation was detected in 62% of light chains, and 69% showed microheterogeneity of charge. There was no clear association between the pI, sialylation, or polymerisation and the presence of renal failure, as assessed by serum creatinine concentrations. Light chains with pI spectrotype in the basic range, however, were found to produce more proximal tubular dysfunction than acidic light chains.

The elimination of alpha 2-macroglobulin complexes from the arthritic joint. An experimental study in dogs.

The polyvalent protease inhibitor, alpha 2-macroglobulin, may counteract a protease-mediated rheumatoid joint destruction. The elimination of the complexed inhibitor from joints was analysed in inflamed and noninflamed conditions of the knee joints in dogs. The arthritis was immunologically induced. The fate of intra-articularly injected radioactive alpha 2-macroglobulin complexes was studied by external measurements, analyses of blood, lymph and urine, and by autoradiographic and immunohistologic methods. The results indicate that the elimination of complexes was accelerated by inflammation and joint movements with a half-life shorter than 2 hours in acute arthritis. In addition to absorption into the synovial membrane and degradation in macrophage-like cells, the process of clearance included elimination of complexes via the blood and the lymph capillaries of the joint and subsequent degradation in cells belonging to the reticuloendothelial system in the lymph nodes and the liver. The degradation products were excreted in increasing amounts in the urine. Referring to the earlier recognized high degree of saturation of synovial fluid alpha 2-macroglobulin in rheumatoid arthritis, the finding of a rapid articular clearance of alpha 2-macroglobulin complexes suggests a pronounced release of endoproteases under clinical conditions.

Qualitative analysis of proteinuria associated with bladder cancer.

In the present investigation molecular components associated with the urines from bladder cancer patients and normal individuals are identified. Polyacrylamide gels of urines from bladder cancer, bladder papilloma, and normal individual exhibit clear differences in banding patterns. Urine from bladder cancer patients shows gels with increased quantities of low (less than 100,000) and increased and additional high (less than 100,000) molecular weight proteins when compared to gels with urine from papilloma and normal individuals. In order to localize and identify proteins in the urine from bladder cancer, papilloma, and normal individuals, proteins were separately fractionated on Sephadex G-200 columns and each elution fraction was reacted on Ouchterlony gel diffusion against various specific antisera. The qualitative analysis of proteins in urine from bladder cancer patients is discussed in relation to their molecular weight distribution in the Sephadex G-200 profiles and their possible role in tumor host relationships.

New approach to evaluation of proteinuric states.

We evaluated the use of immunonephelometric methods for measuring specific urinary proteins. Using a nephelometer to detect light scattering (angle, 31 degrees), we measured some proteins immunonephelometrically in serum and aliquots of 24-h urines from 50 apparently healthy children, ages 2-17 years. The mean urinary excretion rate (mg/24h) and the range of values was: for albumin 5.5 (range, 0-13.3), for transferrin 0.5 (0-1.9, for IgG 3.3 0-12), and for alpha 2-macroglobulin 0.6 (0-2.3). Direct comparison of the values for pathological urines with those for a reference population may offer more meaningful information concerning the integrity of the glomerular basement membrane than is provided by protein selectivity indices, and measuring a plasma protein such as albumin in urine may better define pathological proteinuria.