Jessica Zuin

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

Abstract 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. Clin Chem Lab Med 2010;48:217–23.

Increased antiprotease activity of the SERPINB3 polymorphic variant SCCA-PD

SERPINB3 has been found in chronic liver damage and hepatocellular carcinoma, but not in normal liver. By direct mRNA sequencing, a new SERPINB3 polymorphism (SCCA-PD) has been identified, presenting the substitution Gly351Ala in the reactive center loop of the protein. The prevalence of the SCCA-PD isoform has been found to be significantly higher in patients with cirrhosis than in patients with chronic liver disease and in normal subjects. The aim of this study was to investigate the biological and functional activity of SERPINB3 isoforms using in vitro models. HepG2 and Huh7 cells lines were transfected with plasmid vectors containing wild-type SERPINB3 or its polymorphic variant SCCA-PD and their expression at transcriptional and protein level was determined. To assess the functional activity, both recombinant proteins were produced and kinetic analysis was carried out using papain and cathepsin-L as target proteases. In addition, the inhibition of JNK kinase activity by SERPINB3 isoforms was assessed. The crystal structure of wild-type SERPINB3 at 2.7 Å resolution was used for preparation of refined 3D models of the two isoforms. The results showed that transcriptional activity and protein expression of the two isoforms were similar in both transfected cell lines. Both SERPINB3 preparations exerted a dose-dependent protease inhibitory activity, but the effect of SCCA-PD was higher than that of the wild-type isoform. This result was supported by 3D modelling, where increased hydrophobic profile of the SCCA-PD isoform, introduced by the G351A mutation, was detected. In addition, at high protein concentration, SCCA-PD revealed a 16% higher inhibitory effect on c-Jun phosphorylation by JNK1, compared with wild-type SERPINB3. In conclusion, the single amino acid substitution in the SERPINB3 reactive site loop improves the functional activity of SCCA-PD isoform. This different antiprotease activity might favor disease progression in patients carrying this polymorphism.

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.

Combination of Biomarkers-IGM by Logistic Regression Improves Diagnostic Accuracy in Hepatocellular Carcinoma

Background and aim Circulating immune complexes of alpha-fetoprotein (AFP) and SCCA (squamous cell carcinoma antigen) with IgMs (AFP-IgM and SCCA-IgM, respectively) represent a promising new class of serum markers with diagnostic and prognostic value in the management of patients with hepatocellular carcinoma (HCC). The enhancement of diagnostic indexes for cancer detection achieved with combination of biomarkers by linear logistic regression has been demonstrated in several simulation studies on multiple diagnostic assays. The aim of the study was to evaluate the improvement of the diagnostic accuracy by combination of SCCA-IgM and AFP-IgM compared with the diagnostic accuracy of free AFP in sera of patients with HCC and cirrhosis. Patients and methods Serum samples from 81 patients with HCC (mean age ± SD: 63 ± 8 years; male patients 73%; HCV infected 52%; without any therapeutic treatment 50%) and 82 patients with cirrhosis (mean age ± SD: 53 ± 9 years; male patients 66%; HCV infected 68%) were collected. The logistic regression parameters were calculated on a previously published data set of 50 patients with HCC and 50 patients with cirrhosis, where the distribution of serum levels of SCCA-IgM and AFP-IgM in HCC patients was significantly different from that in patients with cirrhosis (Mann-Whitney U test, p<0.01). Serum levels of SCCA-IgM and AFP-IgM were assessed in parallel using the Hepa-IC and Hepa AFP-IC kits and AFP was determined by an automatic analyzer (ADVIA Centaur®, Siemens Diagnostics, Italy). Results The patients were stratified according to sex (male), HCV infection (positive) and therapeutic treatment (negative) to apply a linear logistic regression model using regression parameters calculated from a data set of a reference population. A subgroup of 30 patients with HCC and 41 patients with cirrhosis was analyzed. The diagnostic accuracy measured as the area under the ROC curve (AUC) for the SCCA-IgM and AFP-IgM assays was roughly the same, with a weak supremacy of AFP-IgM (AUC = 0.62), and was comparable with that of free AFP (AUC = 0.64). The gain in diagnostic accuracy achieved with the combination of SCCA-IgM and AFP-IgM levels by linear logistic regression was 14% (AUC = 0.71) compared with the diagnostic accuracy of AFP-IgM and 11% compared with that of free AFP. With the estimation of the partial AUC (pAUC0.3), an alternative measurement of AUC that takes into consideration only specificity rates with clinical relevance (specificity >70%), the highest improvement in accuracy for HCC detection was obtained, with an increase of up to 62% compared to pAUC0.3 of AFP-IgM and up to 23% compared to pAUC0.3 of free AFP. Conclusion The results demonstrate that the combination by linear logistic regression of biomarkers-IgM immune complexes improves the diagnostic accuracy for HCC detection using regression parameters calculated in a reference population with defined clinical characteristics. These results support the usefulness of devices based on a panel of non-overlapping biomarkers-IgM complexes, such as multi-marker biochips, increasing the sensitivity and maintaining a high specificity for HCC diagnosis compared with conventional single-marker assays.

Osteopontin-Igm as a Marker for the Diagnosis of Hepatocellular Carcinoma

Introduction Biomarkers for early hepatocellular carcinoma (HCC) detection are still a clinical need. Recent data indicate that cancer-associated antigens lead to the formation of circulating IgM-linked immune complexes as the result of natural IgM production by the innate immune response. The SCCA-IgM immune complex has already been described for primary liver cancer detection. Osteopontin (OPN) is a member of the SIBLING family of proteins recently shown to be related to tumorigenesis, progression and metastasis in various cancer types. Aim To evaluate the serum levels of the OPN-IgM complex in comparison to the SCCA-IgM complex in patients with HCC. Patients A total of 256 patients were analyzed including 151 patients with HCC (M/F 115/36; mean age ± SD: 67 ± 12 years) and 106 patients with cirrhosis (M/F 68/38; mean age ± SD: 62 ± 12 years). HCC, tested before any therapeutic approach, was mainly of viral etiology (58% HCV, 11% HBV), while alcohol abuse (22%) was the main risk factor for the remaining cases. A similar distribution was found in patients with cirrhosis (46% HCV, 15% HBV, 38% alcohol). Methods Serum levels of OPN-IgM were measured using a homemade ELISA assay with a polyclonal anti-human OPN antibody (Biodesign Int, USA). SCCA-IgM was detected in serum using an ELISA assay kit (Hepa-IC, Xeptagen). Results OPN-IgM was positive in 64/151 (42%) patients with HCC and in 45/106 (42%) patients with cirrhosis (specificity 58%), while the reference biomarker SCCA-IgM showed 35% sensitivity (49/139) and 71 % specificity. When patients were stratified on the basis of etiology, the sensitivity values for OPN-IgM and SCCA-IgM were 51% vs 44% in HCV patients, and 36% vs 23% in the other patients, while the specificity was lower for OPN-IgM (55% vs 69% in HCV patients; 62% vs 72% in the other patients). Combination of the two biomarkers resulted in an increase in sensitivity to 63% for viral etiology and to 40% for nonviral etiology. Conclusion Different IgM-linked biomarkers are detectable in primary liver cancer. OPN-IgM and SCCA-IgM showed a similar behavior, while the combination of these biomarkers increased the diagnostic performance for primary liver cancer.

The Association of PSA-IGM and Total PSA Improves the Diagnosis of Prostate Cancer

Background and aim The specific causes of prostate cancer (Pca) are unknown but the main risk factors of tumor development are associated with age, genetic factors, ethnicity, diet and lifestyle. Prostate cancer is rare in men under 45 years of age, but becomes more common with advancing age. The main diagnostic tools for demonstrating the presence of PCa include digital rectal examination, transrectal ultrasonography, and serum measurement of prostate specific antigen (PSA) followed by prostate biopsy for confirmation of the diagnosis. While the measurement of PSA levels has revolutionized the diagnosis of PCa, it has also increased its overdiagnosis due to the poor diagnostic accuracy. Scientific evidence indicates that biomarkers for different types of cancer such as liver and colorectal cancer circulate in the blood associated with immunoglobulin M (IgM) to form complexes that allow a better diagnosis in comparison to circulating free biomarkers. In prostate cancer it has been demonstrated that testing for serum levels of the PSA-IgM immune complex improves the diagnostic performance of total PSA. The aim of this study was to evaluate the diagnostic accuracy of PSA-IgM compared to total PSA for the selection of patients to be submitted to transrectal ultrasound-guided prostate biopsy. Patients and methods Serum samples from 67 male patients, 33 affected by PCa with a Gleason score from 5 to 7, and 34 affected by benign prostate hypertrophy (BPH), were collected by the Department of Urology of the Spedali Civili of Brescia. The samples were immediately snap frozen at −80°C. Serum levels of PSA-IgM were assessed using Prostate-IC (Xeptagen, Italy) while PSA levels were determined with the Immulite 2000 of Medical Systems S.p.A. Results Patients were stratified into 2 groups according to age; the first group consisted of 24 patients with PCa and 20 with BPH aged between 60 and 70 years and the second group consisted of 9 patients with PCa and 14 with BPH aged between 70 and 80 years. Serum levels of PSA and PSA-IgM were analyzed in the 2 groups using cutoff values of 4 ng/mL for PSA and 145 AU/mL for PSA-IgM. In the first group, 1 8 of 24 PCa patients were positive for PSA (75% sensitivity) with a specificity of 50% (10 of 20 BPH patients), and 1 0 of 24 PCa patients were positive with the PSA-IgM assay (42% sensitivity), which had a higher specificity (70%; 6 of 20 BPH patients). The combination of both biomarkers resulted in a sensitivity of 38% (9 of 24 patients with PCa) but showed a significant improvement in specificity up to 90%, since 18 of 20 patients with BPH were negative for at least one test. In the second group of patients aged 70 to 80 years, the PSA test had a sensitivity of 67% (6/9 PCa patients) and a specificity of 78% (3/14 BPH patients) compared with a sensitivity of 44% for the PSA-IgM test (4/9 PCa patients) with a specificity of 71% (4/14 BPH patients). The combination of PSA and PSA-IgM had a sensitivity of 30% (3/9) but the highest specificity (93%, 13/14 BPH patients). Conclusion The results of the study demonstrate the diagnostic value of the PSA-IgM assay compared with the total PSA test. The combination of PSA-IgM with total PSA was the best approach to reduce the number of false-positive results, thus improving the diagnosis of prostate cancer.

Evaluation of the Analytical Specificity of SCCA-IGM Assay for Monitoring Patients with Cirrhosis

Background and aim An increasing number of clinical data suggests the importance of the assessment of serum levels of the squamous cell carcinoma antigen (SCCA)-lgM immune complex for the diagnosis of hepatocellular carcinoma (HCC). In addition, monitoring of SCCA-IgM immune complexes has been described as a useful prognostic approach in patients with cirrhosis since the progressive increase of SCCA-IgM over time is associated with a higher risk of HCC development. Because other assays in patients with cirrhosis have been affected by interfering IgMs, the aim of the present study was to assess the specificity of SCCA-IgM reactivity in cirrhotic patients by evaluating SCCA-IgM detection dependence on different capturing phases and by measuring the recovery of SCCA-IgM reactivity after serum fractionation. Patients and methods Serum samples from 82 patients with cirrhosis (M/F ratio 3/1; mean age ± SD: 56 ± 9 years) were collected at the Liver Unit of the Department of Clinical and Experimental Medicine, University of Padua, according to the approved institutional procedures. Serum levels of SCCA-IgM were measured using two different ELISA tests: the reference assay based on a rabbit oligoclonal anti-human SCCA antibody (Hepa IC, Xeptagen, Italy) and a dedicated ELISA with a mouse monoclonal anti-SCCA as capture antibody. Results Serum levels of SCCA-IgM measured with the reference assay (median value 87 AU/mL) were higher than levels measured with the mouse monoclonal test (median value 78 AU/mL), but the differences were not statistically significant (Mann-Whitney U test, p>0.05). When SCCA-IgM levels measured with both tests were compared, a linear correlation was found (r = 0.77, p < 0.001). An in silico analysis using available structural data of SCCA showed the presence of up to three putative antigenic sites localized on the SCCA surface, thus providing evidence that the test with the mouse monoclonal anti-SCCA antibody could underestimate the total circulating immune complexes due to the steric hindrance of the enormous mass of IgM (900 kDa) that could mask on the SCCA (45 kDa) surface the binding site recognized by the monoclonal antibody. To show that the SCCA-IgM assay was not affected by interfering IgMs, we measured the recovery of SCCA-IgM reactivity after serum fractionation of ten of the most reactive samples in both assays. Total recovery of SCCA-IgM reactivity was obtained with both assays in the fractions corresponding to components with higher molecular weight than IgM and SCCA (>2000 kDa). Conclusions The results of this study indicate that the reactivity measured in cirrhotic patients is related only to SCCA-IgM immune complexes and is not affected by other serum components, supporting the analytical specificity of the SCCA-IgM assay and validating the importance of SCCA-IgM as a risk biomarker for HCC development.

Circulating Survivin-IGM is a Novel Candidate Biomarker of Cirrhosis and Increases with Child Score in Patients Affected by Liver Diseases

Background Survivin, also known as BIRC5, is the smallest member of the mammalian IAP family and is a well recognized inhibitor of apoptosis with an important role in cell-cycle regulation. It is detected in fetal and neoplastic adult tissue, but not in normal tissues. Survivin acts on cancer promotion not only by the inhibition of apoptosis but also by acceleration of the proliferative activity of cancer cells and several papers have suggested the involvement of this protein in hepatocarcino-genesis. It has been reported that the detection of HCC biomarkers circulating as IgM immune complexes (ICs) improved the diagnosis and prognosis of HCC. The aim of this study was to compare the expression of survivin as an IgM immune complex in serum from healthy controls and of patients with cirrhosis and patients with HCC to identify a novel biomarker for the monitoring of liver diseases. Methods Serum levels of survivin-IgM from 1 97 individuals, including 39 healthy subjects, 94 patients with cirrhosis and 64 with HCC, were measured by ELISA and the relationship with clinical parameters was evaluated. Results Survivin-IgM was almost undetectable in sera from healthy subjects, high in patients with cirrhosis, and moderately lower in patients with HCC. The survivin-IgM assay was positive in 62 of 94 patients with cirrhosis (66%) and in 28 of 64 patients with HCC (43.7%) using a cut-off of 264.89 AU/mL (specificity of 94%). Statistical analysis showed that IC values were significantly different between groups (cirrhosis versus healthy control group, p<0.001; HCC versus cirrhosis group, p<0.001). Circulating survivin-IgM ICs in patients with HCC were lower than in patients with cirrhosis; in fact, the statistical significance in comparison with healthy subjects was lost. On the other hand, the concentration of circulating survivin-IgM ICs was found to increase with progression of Child score. Conclusions The high expression of survivin-IgM in sera from patients with cirrhosis and the values of sensitivity indicate that survivin-IgM could be a novel candidate biomarker for cirrhotic disease. Furthermore, the increase in ICs with the progression of Child score seems to promote the survivin-IgM immune complex as marker of liver failure. Survivin-IgM was found to be lower in sera from HCC patients. Follow-up studies are in progress to monitor patients with cirrhosis and to validate the association of the downregulation of this marker with progression towards hepatocellular carcinoma.

The Usefulness of the Combination of Free CA 15.3 and CA 15.3-IGM Complexes for Breast Cancer Diagnosis

Background Several studies have shown that the main circulating biomarkers of liver and colorectal cancer can be detected in the bloodstream and are also associated with immunoglobulin M to form stable complexes. These immune complexes show increased capacity of discrimination between cancer patients and healthy controls if combined with the free biomarker form. Within the context of the Project FIRB 2003 - Nanosized Cancer Polymarker Biochip - we wanted to investigate if IgM complexes have importance also in breast cancer. We focused our study on the immune complexes between IgM and CA 15.3 because free CA 15.3 is the most commonly used breast cancer biomarker in clinical practice. However, this biomarker alone lacks satisfactory sensitivity especially in early cancer detection. Aim The aim of our study was to assess the occurrence of immune complexes between CA 15.3 and IgM in sera from patients with primary breast cancer and in sera from healthy controls to evaluate its putative diagnostic value compared with the diagnostic value of free CA 15.3. Methods A total of 130 serum samples were obtained from 56 healthy women (mean age±SD, 45±8.23 years) and 74 women with stage l and II breast cancer (mean age±SD, 59±13.6 years) before any treatment, either surgical or chemo-therapeutic. Serum samples were collected, aliquoted and stored at-80°C in the centralized biobank of the project according to very stringent standard operating procedures (SOPs) distributed by the coordinating unit. To evaluate the presence of CA 15.3-lgM immune complexes, we developed and validated a novel enzyme-linked immunosorbent assay (ELISA) with a polyclonal rabbit anti-human CA 15.3 antibody (Abcam) as the catcher antibody. CA 15.3-lgM was detected with peroxidase-conjugated anti-human IgM and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide as substrate (Sigma Aldrich, Italy). The levels of CA 15.3-lgM were expressed in arbitrary units per mL (AU/mL) by interpolation on a calibration curve obtained by serial dilution of a reference calibrator purified by gel filtration chromatography from a pool of serum samples with high levels of CA 15.3-lgM. Serum levels of free CA 15.3 were assessed in parallel on each sample using an automated immunoassay system (ADVIA Centaur-Siemens Diagnostics) and expressed in U/mL. Results To discriminate between cancer patients and healthy controls, we used as cutoff values 31.5 U/mL for free CA 15.3 (corresponding to the cutoff used in the clinical routine) and 794 AU/mL for CA 15.3-lgM (representing the 95th percentile of the distribution of serum levels of CA 15.3-lgM in healthy controls). By using these cutoff values, we obtained a sensitivity of 1 0% (7/74 cases) and a specificity of 95% (53/56 controls) for CA 15.3-lgM. The sensitivity and specificity of free CA15.3 were 7% (5/74 cases) and 1 00% (56/56 controls), respectively. Interestingly, the serum levels of the two biomarkers did not overlap, so their combination at 95% specificity identified 12/74 cases (16.2%). When we took a cutoff of 22 U/ mL for free CA15.3 (the 95th percentile of its distribution in healthy controls), we calculated a sensitivity of 26% (1 9/74 cases) and a specificity of 95% (53/56 controls); its combination with CA 15.3-lgM had a sensitivity of 34% (25/74 cases) and a specificity of 90% (50/56 controls). Conclusions These results demonstrate for the first time the presence of CA 15.3-lgM in the bloodstream of patients with breast cancer. In addition, our data suggest that CA 15.3-lgM is a complementary serological marker to free CA 15.3 and the combination of these biomarkers could improve the diagnosis of breast cancer.