Photoplethysmography (PPG)-determined heart rate variability (HRV) and extracellular water (ECW) in the evaluation of chronic stress and inflammation.

BACKGROUND: Heart rate variability (HRV) is the physiological variation in the time interval between consecutive heartbeats. Extracellular water (ECW) is the aqueous compartment surrounding cells and has been used as an inflammation index. Medically unexplained symptoms (MUS) refer to persistent psychosomatic bodily complaints, and their presence may be employed as a clinical index of chronic stress and inflammation, useful in distinguishing suffering patients from healthy subjects. AIM: To evaluate the clinical performance of SDNN (standard deviation of intracardiac beat time interval of normal sinus beats) and RMSSD (square root of the mean squared differences of successive NN intervals) as indices of HRV, and their correlation with ECW and MUS. PATIENTS AND METHODS: A large multicenter retrospective study was conducted in 37 Italian medical practices in Caucasian men and women aged 20 to 70 years. SDNN and RMSSD were measured with the PPG Stress Flow® device (BioTekna, Italy), while ECW was determined with the BIA-ACC® device (BioTekna, Italy). All subjects filled in a MUS® questionnaire with 19 "nonspecific" symptom questions. The study sample was stratified by decade of age into five groups. RESULTS: Data from 9246 subjects comprising 3127 males and 6119 females, with a median age of 47 years, were analyzed. HRV index SDNN and RMSSD distributions in the entire sample and in each of the five age groups were significantly greater in subjects with a limited number of MUS (0-5) than in subjects with six or more symptoms, while both distributions correlated negatively with ECW. CONCLUSION: SDNN and RMSSD and ECW were predictors of MUS and were successfully used to objectively evaluate chronic stress and inflammation.

Current evidence and future perspectives about the role of iXip® in the diagnosis of prostate cancer.

iXip® (Immune CompleX Predictive Index, Xeptagen, Venice, Italy) is a diagnostic tool which biological bases ground on PSA-IgM complexes. An algorithm merges the data of PSA-IgM and serum total PSA dosage, prostate volume and patient's age, providing as output a numerical value that correlates with the risk of finding prostate cancer (PCa) at biopsy. The present paper reviews the available evidence and explores future perspective on iXip. A few studies consistently showed that iXip offers better diagnostic accuracy in the diagnosis of PCa than every single parameter composing the index. In detail, for values of iXip below 20% prostatic biopsies were invariably negative, between 20% and 30% only one out of 10 patients had cancer, generally Gleason Score 6, whereas for iXiP>30% the detection rate raised up to 35% and comprised the majority of Gleason score >6 cancers. The PROXIMA study is an ongoing prospective trial that should assess the predictive ability of iXip towards the presence of a clinically significant PCa defined at radical prostatectomy, accounting for clinical, multiparametric magnetic resonance and bioptic data. Preliminary data showed that for iXip values <20% prostatic biopsy could be safely omitted and that the diagnosis of Gleason Score >6 PCa is unlikely for values below 30%.

Development of a novel diagnostic algorithm to predict NASH in HCV-positive patients.

Non-alcoholic steato-hepatitis (NASH) is a severe disease characterised by liver inflammation and progressive hepatic fibrosis, which may progress to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests that in hepatitis C virus patients steatosis and NASH are associated with faster fibrosis progression and hepatocellular carcinoma. A safe and reliable non-invasive diagnostic method to detect NASH at its early stages is still needed to prevent progression of the disease. We prospectively enrolled 91 hepatitis C virus-positive patients with histologically proven chronic liver disease: 77 patients were included in our study; of these, 10 had NASH. For each patient, various clinical and serological variables were collected. Different algorithms combining squamous cell carcinoma antigen-immunoglobulin-M (SCCA-IgM) levels with other common clinical data were created to provide the probability of having NASH. Our analysis revealed a statistically significant correlation between the histological presence of NASH and SCCA-IgM, insulin, homeostasis model assessment, haemoglobin, high-density lipoprotein and ferritin levels, and smoke. Compared to the use of a single marker, algorithms that combined four, six or seven variables identified NASH with higher accuracy. The best diagnostic performance was obtained with the logistic regression combination, which included all seven variables correlated with NASH. The combination of SCCA-IgM with common clinical data shows promising diagnostic performance for the detection of NASH in hepatitis C virus patients.

Characterization of SCCA-IgM as a biomarker of liver disease in an Asian cohort of patients.

Viral hepatitis infection is a major global issue and a leading cause of liver disease and associated deaths. Over time, patients infected with hepatitis B (HBV) or C virus (HCV) develop cirrhosis and, eventually, hepatocellular carcinoma (HCC). For this reason, they need to be constantly monitored. Current Asian guidelines recommend the determination of serum alpha-fetoprotein (AFP) together with liver ultrasounds every six months to detect HCC nodules. However, both methods have several limitations, and other biomarkers have been studied for monitoring cirrhosis, including SCCA-IgM, an immune-complex formed by Squamous Cell Carcinoma Antigen and IgM. To date, SCCA-IgM has been validated as a novel biomarker for liver diseases only in European populations. The aim of our study was to analyze SCCA-IgM as a biomarker to monitor cirrhosis evolution in an Asian cohort of patients and to compare its performance to that of AFP. We analyzed the concentration of AFP and SCCA-IgM in serum samples obtained from a group of Asian adult patients with cirrhosis or HCC and a control group of patients admitted for gastrointestinal disorders. In untreated patients and similarly to AFP, SCCA-IgM levels were significantly higher in patients with cirrhosis compared to those with HCC. In addition, SCCA-IgM, but not AFP serological levels, were significantly lower in HCC patients who were treated with surgical resection compared to those who received a different therapy.

Clinical evaluation of the iXip index to reduce prostate re-biopsies.

BACKGROUND: Prostate biopsy is the gold standard for prostate cancer (PCa) diagnosis, but it's invasive and associated with adverse events. Novel reliable tumor biomarkers and accurate non-invasive tests are required to avoid biopsies. The immune complex PSA-IgM is a new marker for PCa, and it has been included in an algorithm to generate the diagnostic index iXip, which determines the probability of having PCa. In this study we evaluated the ability of iXip to reduce the number of repeat biopsies in patients with a previous negative biopsy and suspicious for PCa. PATIENTS AND METHODS: 219 patients referred for prostate rebiopsy were included in the study. Each patient underwent a trans-rectal ultrasound-guided prostate biopsy and prostate volume examination. Blood samples were collected before any prostatic manipulation to determine the serological levels of PSA-IgM and PSA. The iXip index was calculated as previously reported using an online calculator. RESULTS: iXip values in patients with a positive biopsy were significantly higher than the ones observed in negative patients (p-value = 0.001). Based on iXip values, patients were divided in five risk groups: those with iXip < 0.2 had 0% probability of having PCa. High values of the Gleason score (≥7) were observed mostly in patients with iXip 0.3-0.8. CONCLUSION: Our preliminary results show that iXip identifies a sub-group of patients who can safely avoid rebiopsy because they do not have PCa. The index is a promising tool that could reduce the number of unnecessary prostate biopsies and the relative clinical complications and expenses.

Circulating SCCA-IgM complex is a useful biomarker to predict the outcome of therapy in hepatocellular carcinoma patients.

INTRODUCTION: Hepatocellular carcinoma (HCC) develops in about 3-4% of cirrhotic patients every year. The squamous cell carcinoma antigen (SCCA) has been found elevated in liver cancer specimens by immunohistochemistry, and detected in complex with IgM (SCCA-IgM) in the serum of patients with HCC. The aim of this study was to evaluate the ability of serological SCCA-IgM levels to predict the efficacy of HCC therapy. MATERIALS AND METHODS: From April 2012 to April 2014, 131 patients with a new diagnosis of HCC were enrolled. The HCC diagnosis was made according to the EASL guidelines. The patients were staged and treated according to the BCLC Staging System: BCLC stages A and B were treated with locoregional therapy, and BCLC stage C was treated with Sorafenib. Response to therapy was evaluated according to the mRECIST criteria. Serum SCCA-IgM levels were determined by a commercially available ELISA kit at basal time (T0) and after one month of treatment (T1). RESULTS: At baseline and one month into therapy, SCCA-IgM levels were significantly lower (p value <.05) in patients who responded to therapy compared to those who did not respond (median SCCA-IgM level [25th + 75th percentile] at T0:115.1 AU/mL [50.0 + 174.4] vs. 149.1 AU/mL [111.3 + 198.8]; median SCCA-IgM level [25th + 75th percentile] at T1: 113.4 AU/mL [50.0 + 194.2] vs. 170.6 AU/mL [111.7 + 344.2]). CONCLUSION: Our study suggests that the SCCA-IgM determination could be helpful in predicting the response to therapy in patients with HCC.

Lectin-based lateral flow assay: proof-of-concept.

Lateral flow assays (LFAs) enable the simple and rapid detection and quantification of analytes and is popular for point-of-care (PoC), point-of-use and outdoor testing applications. LFAs typically depend on antibody or nucleic acid based recognition. We present the innovative concept of a LFA using lectins in the role of the biorecognition element. Lectins are a special kind of glycan-binding protein and the lectin-based LFA herein described was developed for the determination of the glycosylation of free prostate specific antigen (PSA). PSA is routinely used as a biomarker of prostate cancer (PCa) and the glycosylation status of PSA is a more specific marker of disease progress than only the PSA level. Using the lectin-based LFA we were able to detect α-2,6 sialic acid present in fPSA using Sambucus nigra (SNA) lectin. As a negative control, we employed Maackia amurensis lectin II (MAA II) which specifically binds α-2,3 sialic acid. The novel approach presented here can be applied to a wide range of biomarkers that have a significant impact on clinical diagnosis and prognosis, providing an alternative to standard lectin-based assays. The assay uses commercial components and is easily performed by applying a sample to the sampling pad on the lectin-based LFA strip, with results obtained within 10 minutes.

Lateral flow assays.

Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored.

Squamous cell carcinoma antigen-IgM is associated with hepatocellular carcinoma in patients with cirrhosis: A prospective study.

BACKGROUND: Squamous cell carcinoma antigen (SCCA)-IgM complex has been described as a promising tool to identify patients with progressive liver disease at higher risk of hepatocellular carcinoma (HCC) development in retrospective studies. AIM: To assess the clinical value of this biomarker in patients with cirrhosis in a prospective study. METHODS: Patients with overt cirrhosis were prospectively evaluated at 6-month intervals for HCC development and decompensation with clinical examination, liver ultrasound, α-fetoprotein measurement. SCCA-IgM was measured in serum by immunoenzymatic assay. Median follow-up duration was 52 months (range 12-68 months). RESULTS: 70 patients (26% male; mean age 56±10 years) were enrolled. The main aetiological factors were alcohol (44%) and hepatitis C (34%). Baseline values of SCCA-IgM were significantly higher in patients who developed HCC. Positivity of the biomarker at baseline was associated with a significantly shorter HCC-free survival, while α-fetoprotein (cut off >20 ng/ml) was not significant. SCCA-IgM positivity and hepatitis C were significant prognostic factors for HCC development. The biomarker was not associated with the development of clinical complications of cirrhosis. CONCLUSION: This prospective study demonstrates that in patients with cirrhosis SCCA-IgM is associated with HCC development and may be useful for clinical management of cirrhotic patients at higher risk of HCC development.

Clinical applications of squamous cell carcinoma antigen-immunoglobulins M to monitor chronic hepatitis C.

Hepatitis C virus (HCV) is the main cause of chronic liver disease and cirrhosis in Western countries. Over time, the majority of cirrhotic patients develop hepatocellular carcinoma (HCC), one of the most common fatal cancers worldwide - fourth for incidence rate. A high public health priority need is the development of biomarkers to screen for liver disease progression and for early diagnosis of HCC development, particularly in the high risk population represented by HCV-positive patients with cirrhosis. Several studies have shown that serological determination of a novel biomarker, squamous cell carcinoma antigen-immunoglobulins M (SCCA-IgM), might be useful to identify patients with progressive liver disease. In the initial part of this review we summarize the main clinical studies that have investigated this new circulating biomarker on HCV-infected patients, providing evidence that in chronic hepatitis C SCCA-IgM may be used to monitor progression of liver disease, and also to assess the virological response to antiviral treatment. In the last part of this review we address other, not less important, clinical applications of this biomarker in hepatology.

A novel algorithm for the prediction of prostate cancer in clinically suspected patients.

BACKGROUND: Prostate cancer (PCa) represents the most common solid tumor affecting men and its early detection remains the best approach to improve survival rates. The assessment of serum levels of PSA is currently used for PCa screening but the low specificity of the test results in a high number of false positives. Other forms of PSA may be detected in the bloodstream including PSA associated with immunoglobulin M (PSA-IgM) which, alone or combined with PSA, has shown diagnostic accuracy for PCa. OBJECTIVES: The aim of the study is to improve the diagnostic accuracy of PSA-IgM by developing a multivariable model which includes serum biomarkers and routine diagnostic parameters to obtain a predictive index useful in the post-screening clinical practice. PATIENTS AND METHODS: One hundred sixty male patients with clinical suspect of PCa underwent a trans-rectal ultrasound guided first prostate biopsy with a standardized sampling scheme. To generate the model, we assessed the presence of PSA and PSA-IgM complexes in sera of patients and the prostate volume of each patient. A novel predictive probability for PCa (iXip) was obtained combining non-overlapping biomarkers normalized with diagnostic parameters. RESULTS: The study population included 49 patients with PCa diagnosed at biopsy and 111 controls in which prostate biopsy showed the presence of benign prostatic hyperplasia, inflammation, atypical small acinar proliferation or high-grade prostatic intraepithelial neoplasia. The iXip values for patients with PCa (mean ± SD=0.467 ± 0.160) were significantly higher (p-value < 0.001) than control subjects (mean ± SD=0.314 ± 0.098) and the iXip AUC (0.787) was significantly greater (p-value < 0.001) than the AUCs of each biomarker. CONCLUSIONS: iXip shows a significant increase in diagnostic performance compared to PSA and PSA-IgM and its post-screening use may facilitate decision-making in recommending for biopsy clinically suspected patients.

Synthesis and in vitro study of novel neuraminidase inhibitors against avian influenza virus.

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC(50) of 14.6±3.0nM (oseltamivir 25±4nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC(50) of 0.1±0.03nM (oseltamivir 0.2±0.02nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.

A screening assay for neuraminidase inhibitors using neuraminidases N1 and N3 from a baculovirus expression system.

CONTEXT: Development of inexpensive and safe enzymatic assays to screen for putative neuraminidase inhibitors. OBJECTIVE: Validate the use of recombinant neuraminidase expressed in baculovirus located on the viral surface capsule to develop a neuraminidase inhibitor screening assay. MATERIALS AND METHODS: Recombinant baculovirus particles displaying neuraminidase N1 and N3 were used as enzyme sources. The assay set-up required the use of 2'-(4-methylumbelliferyl)-α-D-acetyl neuraminic acid as substrate and oseltamivir carboxylate as benchmark inhibitor. RESULTS: The assay was set up in a standard 96-well plate. The within- and between-assay coefficients of variation were, on average, less than 10%. The 50% inhibitory concentration values of the inhibitor were in good agreement with those determined by independent kinetic experiments. DISCUSSION AND CONCLUSIONS: The assay showed satisfactory within- and between-assay repeatability. The obtained results suggest that recombinant baculovirus expressing neuraminidase located on the virus membrane capsule can be used to set up affordable and reliable neuraminidase inhibitors screening assays.

Structural refinement of protein A mimetic peptide.

A novel dendrimeric peptide ligand dubbed D-PAM-Φ was designed to achieve a high capacity for human IgG through the decoration of the D-PAM scaffold. The design criteria based on the introduction of small hydrophobic groups on the D-PAM structure were supported by the recently published solid-state structure of D-PAM complexed to the Fc fragment of a recombinant human IgG1 and by molecular dynamic simulations that provided information on the mode of binding of phenylacetyl-D-PAM (D-PAM-Φ). D-PAM-Φ was immobilised on an activated solid support and compared with the parent D-PAM affinity matrix. The newly obtained affinity sorbent was evaluated for its capacity to selectively capture polyclonal human IgG; the binding capacity was approximately 10 mg/ml, an almost 10-fold enhancement with respect to the D-PAM-functionalised matrices without the specificity of binding being reduced. The new ligand was also effective in the capturing of recombinant humanised IgG1 from a clarified cell culture supernatant. Under a typical laboratory-scale affinity chromatography assembly and preliminarily optimised binding conditions, the affinity purification of humanised IgG1 from culture supernatants rendered the desired product, with purity higher than 90%. The results suggest that the application of the computational approach on the structure of the D-PAM-Fc complex may be very valuable in the development of novel lead molecules for the downstream processing of human or humanised antibodies used in therapy.

The quenching effect of flavonoids on 4-methylumbelliferone, a potential pitfall in fluorimetric neuraminidase inhibition assays.

Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10(-5)), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.

Solid-phase preparation of protein complexes.

Protein-protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin-biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies.

Combinatorial semisynthesis of biomarker-IgM complexes.

Circulating immune complexes formed by tumor antigens and immunoglobulin M (IgM) represent a novel class of biomarkers with diagnostic value for early cancer detection. The quantitative analysis of these immune complexes is achieved by enzyme-linked immunosorbent assay (ELISA) methods using a purified calibrator from samples of patients with cancer. These complexes obtained from samples of human origin are not suitable for cost-effective production processes with high safety standards. Given the ill-defined biomarker/IgM ratio in these complexes, semisynthesis with retention of functional properties is difficult to achieve and may vary widely according to the batch-to-batch heterogeneity of starting biological preparations. Here the authors describe the development of a combinatorial method for defining the optimal reaction conditions for the reproducible semisynthesis of biomarker-IgM complexes by exploiting the biotin-avidin technology. The method relies on screening by ELISA the 3D composition space defined by the combinatorial variation of biotinylated-biomarker, biotinylated-IgM, and avidin concentrations aiming to select those conditions leading to biomarker-IgM complexes with the highest immunoreactivity. The method allows the reproducible synthesis of species with immunoreactivity comparable to that of natural immune complexes and endowed with sufficient stability to be used as calibrators in ELISA.

Experimental validation of specificity of the squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) assay in patients with cirrhosis.

BACKGROUND: Squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) is a useful biomarker for the risk of development of hepatocellular carcinoma (HCC) in patients with cirrhosis due to its progressive increase associated to HCC evolution. In patients with cirrhosis, other assays have been affected by interfering reactivities of IgM. In this study, the analytical specificity of the SCCA-IgM assay was assessed by evaluating SCCA-IgM measurement dependence on different capture phases, and by measuring the recovery of SCCA-IgM reactivity following serum fractionation. METHODS: Serum samples from 82 patients with cirrhosis were analyzed. SCCA-IgM was measured using the reference test (Hepa-IC, Xeptagen, Italy) that is based on rabbit oligoclonal anti-squamous cell carcinoma antigen (SCCA) and a dedicated ELISA with a mouse monoclonal anti-SCCA as the capture antibody. RESULTS: SCCA-IgM concentrations measured with the reference assay (median value=87 AU/mL) were higher than those measured with the mouse monoclonal test (median value=78 AU/mL). However, the differences in the SCCA-IgM distribution were not statistically significant (p>0.05). When SCCA-IgM concentrations measured with both tests were compared, a linear correlation was found (r=0.77, p<0.05). Fractionation of the most reactive sera by gel-filtration chromatography showed that total recovery of SCCA-IgM reactivity was seen only in the fractions corresponding to components with a molecular weight higher than IgM and SCCA (>2000 kDa) with both tests. CONCLUSIONS: The equivalence of both SCCA-IgM assays and the absence of reactivity not related to immune complexes support the analytical specificity of SCCA-IgM measurements. The results validate the assessment of SCCA-IgM for prognostic purposes in patients with cirrhosis.

Detection of prostate-specific antigen coupled to immunoglobulin M in prostate cancer patients.

BACKGROUND: Several reports indicate that the main biomarkers for liver and colorectal cancer circulating in the blood stream associate with immunoglobulin M (IgM) to form stable complexes that show increased diagnostic relevance compared to circulating free biomarkers. METHODS: To further investigate the association between cancer biomarkers and IgM, we assessed the presence of prostate-specific antigen (PSA) as IgM complexes in sera of patients with prostate cancer (PC) or benign prostatic hyperplasia (BPH) in comparison with PSA measurements. RESULTS: PSA-IgM levels were significantly elevated in 40% (20/50) and 12% (6/51) of PC and BPH patients, respectively, compared to 22% (11/50) and 29% (15/51) of PSA positive patients in the same groups. Detection of cancer markedly increased from 22 to 60% by co-determination of both markers (30/50 patients). Significantly elevated levels of PSA-IgM were found in 13 out of 30 patients affected by PC with a PSA value between 4 and 10 ng/mL and only in 4 out of 34 BPH patients in the same PSA range. CONCLUSIONS: The results are the first evidence of the occurrence of PSA-IgM complexes in patients with prostate cancer. The gain achieved in cancer detection by using the combination of PSA and PSA-IgM suggests that PSA-IgM could be a complementary serological marker of prostate cancer.