Inga Laursen

Evaluation of HE4, CA125, risk of ovarian malignancy algorithm (ROMA) and risk of malignancy index (RMI) as diagnostic tools of epithelial ovarian cancer in patients with a pelvic mass

OBJECTIVE Diagnostic factors are needed to improve the currently used serum CA125 and risk of malignancy index (RMI) in differentiating ovarian cancer (OC) from other pelvic masses, thereby achieving precise and fast referral to a tertiary center and correct selection for further diagnostics. The aim was to evaluate serum Human Epididymis protein 4 (HE4) and the risk of ovarian malignancy algorithm (ROMA) for these purposes. METHODS Serum from 1218 patients in the prospective ongoing pelvic mass study was collected prior to diagnosis. The HE4 and CA125 data were registered and evaluated separately and combined in ROMA and compared to RMI. RESULTS 809 benign tumors, 79 borderline ovarian tumors, 252 OC (64 early and 188 late stage), 9 non-epithelial ovarian tumors and 69 non-ovarian cancers were evaluated. Differentiating between OC and benign disease the specificity was 62.2 (CA125), 63.2 (HE4), 76.5 (ROMA) and 81.5 (RMI) at a set sensitivity of 94.4 which corresponds to RMI=200. The areas under the curve (AUC) were 0.854 (CA125), 0.864 (HE4), 0,897 (ROMA) and 0.905 (RMI) for benign vs. early stage OC. For premenopausal benign vs. OC AUC were 0.925 (CA125), 0.905 (HE4), 0.909 (ROMA) and 0.945 (RMI). CONCLUSION HE4 and ROMA helps differentiating OC from other pelvic masses, even in early stage OC. ROMA performs equally well as the ultrasound depending RMI and might be valuable as a first line biomarker for selecting high risk patients for referral to a tertiary center and further diagnostics. Further improvements of HE4 and ROMA in differentiating pelvic masses are still needed, especially regarding premenopausal women.

Human plasma-derived mannose-binding lectin: a phase I safety and pharmacokinetic study

Mannose‐binding lectin (MBL) is an important component of innate immunity that can bind to certain sugar residues on the surface of many types of pathogenic micro‐organisms. On binding, MBL generates opsonic activity mainly through activation of the complement system. Genetically determined MBL deficiency is very common and can be associated with increased susceptibility to a variety of infections, especially in children and immunosuppressed individuals. The potential benefits of MBL reconstitution therapy therefore need to be evaluated. We have carried out a phase I safety and pharmacokinetic study on 20 MBL‐deficient healthy adult volunteers. The MBL was prepared from plasma of nonremunerated, voluntary Danish donors tested and found negative for hepatitis B surface antigen, antibodies to human immunodeficiency virus (HIV) and hepatitis C virus. Each volunteer received a total of 18 mg of MBL in three 6 mg doses given intravenously, once weekly over a period of 3 weeks. The volunteers were closely monitored at the University Hospital in Reykjavik for 8 h after each infusion and daily thereafter for 5 days after each infusion. No adverse clinical or laboratory changes were observed in any of the 20 participants, and frequent measurements did not reveal any signs of infusion‐associated complement activation. No antibodies to MBL, HIV or hepatitis viruses were observed 24 weeks after the last infusion. Serum MBL levels increased up to normal levels (1200–4500 ng/ml) immediately after each infusion, but the half‐life of the infused MBL was highly variable, ranging from 18 to 115 h (mean 69.6). It is concluded that infusion of purified MBL as prepared by Statens Serum Institut (SSI) is safe. However, adults have to be given at least 6 mg twice or thrice weekly for maintaining protective MBL levels assumed to be about 1000 ng/ml.

Low levels of mannose-binding lectin do not affect occurrence of severe infections or duration of fever in acute myeloid leukaemia during remission induction therapy.

Abstract: Purpose: To estimate the clinical significance of low serum concentrations of mannose‐binding lectin (MBL) in patients with acute myeloid leukaemia (AML) during initial cancer chemotherapy. Patients and methods: 80 consecutive, newly diagnosed, and unselected AML patients (age 18–77 yr) undergoing remission induction chemotherapy. The patients were examined for 28 d. Main findings: Low levels of serum MBL (<1000 μg/L) were found in 16/80 patients at diagnosis. This frequency is similar to what is found in the general population. In the remaining 64 patients, MBL concentrations were significantly higher than in controls and showed only a slight rise during the period of antineoplastic chemotherapy with its associated infectious complications. Low levels of MBL did not affect overall survival or morbidity in terms of incidence or duration of fever, or occurrence of septicaemia or pneumonia. Long‐term survival was likewise independent of MBL concentration. Conclusion: MBL levels have no discernible influence on the occurrence or course of infections in AML patients during the initial hospitalisation. The predominant immunodeficiency during this phase is the profound granulocytopenia, which also compromises important effector functions of MBL. The finding in most AML patients of elevated MBL concentrations on admission is most likely because of the role of MBL as an acute phase reactant.

Safety and pharmacokinetics of plasma-derived mannose-binding lectin (MBL) substitution in children with chemotherapy-induced neutropaenia

Mannose-binding lectin (MBL)-deficient children with cancer may benefit from substitution of the innate immune protein MBL during chemotherapy-induced neutropaenia. We determined the safety and pharmacokinetics of MBL substitution in a phase II study in MBL-deficient children. Twelve MBL-deficient children with cancer (aged 0-12 years) received infusions of plasma-derived MBL once, or twice weekly during a chemotherapy-induced neutropaenic episode (range: 1-4 weeks). Four patients participated multiple times. Target levels of 1.0 microg/ml were considered therapeutic. In total, 65 MBL infusions were given. No MBL-related adverse reactions were observed, and the observed trough level was 1.06 microg/ml (range: 0.66-2.05 microg/ml). Pharmacokinetics were not related to age after correction for body weight. The half-life of MBL, for a child of 25 kg, was 36.4h (range: 23.7-66.6h). No anti-MBL antibodies were measured 4 weeks after each MBL course. Substitution therapy with MBL-SSI twice weekly was safe and resulted in trough levels considered protective.

A simple immunoblotting method after separation of proteins in agarose gel

A simple and sensitive method for immunoblotting of proteins after separation in agarose gels is described. It involves transfer of proteins onto nitrocellulose paper simply by diffusion through pressure, a transfer which only takes about 10 min. By this method we have demonstrated the existence of multiple molecular forms of the complement factors C3 and factor B in serum from 2 species, man and chicken, after electrophoretic separation in agarose. We have also demonstrated the usefulness of the method for determining the isoelectric point of proteins after isoelectric focusing in agarose.

First trimester maternal serum pregnancy-specific beta-1-glycoprotein (SP1) as a marker of adverse pregnancy outcome.

To establish the first trimester levels of pregnancy‐specific beta‐1‐glycoprotein (SP1) in pregnancies with adverse outcome. Furthermore, to determine the screening performance for adverse outcome using SP1 alone and in combination with other first trimester markers including proMBP and PAPP‐A.

Mannose-binding lectin (MBL) substitution: recovery of opsonic function in vivo lags behind MBL serum levels.

Mannose-binding lectin (MBL) deficiency is often associated with an increased risk of infection or worse prognosis in immunocompromised patients. MBL substitution in these patients might diminish these risks. We therefore performed an open, uncontrolled safety and pharmacokinetic MBL-substitution study in 12 pediatric oncology patients with chemotherapy-induced neutropenia. Twice weekly MBL infusions with plasma-derived MBL yielded MBL trough levels >1.0 μg/ml. We tested whether MBL substitution in vivo increased MBL-dependent complement activation and opsonophagocytosis of zymosan in vitro. Upon MBL substitution, opsonophagocytosis by control neutrophils increased significantly (p < 0.001) but remained suboptimal, although repeated MBL infusions resulted in improvement over time. The MBL-dependent MBL-associated serine protease (MASP)-mediated complement C3 and C4 activation also showed a suboptimal increase. To explain these results, complement activation was studied in detail. We found that in the presence of normal MASP-2 blood levels, MASP-2 activity (p < 0.0001) was reduced as well as the alternative pathway of complement activation (p < 0.05). This MBL-substitution study demonstrates that plasma-derived MBL infusions increase MBL/MASP-mediated C3 and C4 activation and opsonophagocytosis, but that higher circulating levels of plasma-derived MBL are required to achieve MBL-mediated complement activation comparable to healthy controls. Other patient cohorts should be considered to demonstrate clinical efficacy in phase II/III MBL-substitution studies, because we found a suboptimal recovery of (in vitro) biological activity upon MBL substitution in our neutropenic pediatric oncology cohort.

The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases.

The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan‐binding lectin (MBL) was investigated by solid‐phase binding assays. Calreticulin showed saturable and time‐dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non‐covalent and biphasic with an initial salt‐sensitive phase followed by a more stable salt‐insensitive interaction. For plasma‐derived MBL, known to be complexed with MBL‐associated serine proteases (MASPs), no binding was observed. Interaction of calreticulin with recombinant MBL was fully inhibited by recombinant MASP‐2, MASP‐3 and MAp19, but not by the MASP‐2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co‐receptor/chaperone for both proteins. In conclusion, the potential MBL co‐receptor calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.

Gc globulin (vitamin D‐binding protein) levels: an inhibition ELISA assay for determination of the total concentration of Gc globulin in plasma and serum

Gc globulin, also called vitamin D‐binding protein, is a plasma protein involved in the actin‐scavenger system. In this study, the total Gc globulin concentration in serum or plasma samples was determined using a new, fast, solid‐phase inhibition assay. Included in the study were 228 healthy volunteers (131 M, 97 F), 22 pregnant women, 90 cancer patients and 9 patients with chronic liver disease. Moreover, the degree of complexing with actin was determined in selected samples using crossed immunoelectrophoresis. The Gc globulin level in healthy controls was in the range 176–623 mg/L, showing no age dependency. The median level was found to be significantly higher in women than in men. Gc globulin concentrations were raised during pregnancy, showing a median value of 541 mg/L in the first trimester, and slightly raised to 574 mg/L in the second trimester. Cancer patients showed no changes in Gc globulin level, and there was no sign of increased amounts of complexing with actin. Chronic liver patients showed increased levels of Gc globulin following transplantation, but no signs of complexing with actin. This new solid‐phase inhibition assay is fast, it is a good complement to the existing quantification methods, and it is especially suitable for determination of the Gc globulin status in acute liver patients before and during treatment.

Interaction of C1q with the receptor calreticulin requires a conformational change in C1q.

The interaction between C1q and the chaperone calreticulin was studied under various conditions. When both proteins were present in equal amounts in solution, no interaction could be demonstrated. However, C1q immobilized on a hydrophobic surface, exposed to heat‐treatment or bound to immunoglobulins (Igs) showed a strong, rapid and specific binding of calreticulin. The interaction appeared to be a two‐step process, and the initial phase of interaction was sensitive to high concentrations of salt but not to a physiological salt concentration. The following strong binding was insensitive to salt and extremes of pH but sensitive to strongly denaturing agents (urea and guanidine). The sensitivity to salt during the initial phase of interaction was practically identical to that observed when calreticulin was bound to type V collagen. Binding between C1q and calreticulin could be inhibited by serum amyloid P component and by proteinase K‐digested ovalbumin, and the binding of calreticulin to proteinase K‐digested ovalbumin was shown to be inhibited by C1q. The data indicate that C1q binds stably to the peptide‐binding site of calreticulin and that the initial binding of calreticulin to C1q involves the collagen‐like domain of the C1q molecule. In conclusion, our results suggest calreticulin as a potential receptor for an altered conformation of C1q as occurs during binding to Igs. Thus, the chaperone and protein‐scavenging function of calreticulin may extend from the endoplasmic reticulum to the topologically equivalent cell surface, where it may contribute to the elimination of immune complexes and apoptotic cells.