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A Nanoparticle-Lectin Immunoassay Improves Discrimination of Serum CA125 from Malignant and Benign Sources.

Serum cancer antigen 125 (CA125) [9] is a glycoprotein serum biomarker used to diagnose and monitor epithelial ovarian cancer (EOC), especially its most common and malignant variant high-grade serous ovarian cancer (1). However, increased circulating CA125 is also found in patients suffering from endometriosis, liver disease, and ovarian cysts (2). The generally applied cutoff value of 35 U/mL CA125 does not reliably predict EOC (2), and due to this poor clinical specificity CA125 measurement is not recommended for EOC screening (2). More recently, attempts have been made to combine several tumor markers to increase clinical sensitivity and specificity of EOC detection, but CA125 has remained the preferred biomarker (3-4).

Aberrant glycosylation occurs in many types of human cancers, and glycan structures such as [beta]1-6 branched A-glycans and the Tn antigen (truncated O-glycan) are common in many tumor types (5-7). Carbohydrate epitopes constitute many cancer-associated antigens (8), and detection of cancer-related glycosylation patterns of proteins represents a promising approach for improved cancer detection. Techniques including mass spectrometry, nuclear magnetic resonance, and chromatographic glycoprofiling have enabled analyses of the glycan structures present in cancer cells, sera and tissues (7, 9-11). Such approaches have been used to identify potential glycan markers for prostate (12), breast (13), liver (14), and ovarian cancers (9, 15-16).

CA125 is a large transmembrane mucinlike molecule (MUC16 mucin), with abundant N- and O-glycans (249 potential N-glycosylation and over 3700 O-glycosylation sites) that comprise about 28% of its molecular mass (15, 17). Twenty percent of the sugar moieties in the ovarian cancer cell line, OVCAR3-derived CA125, contains both high mannose type and complex type N-glycan structures, whereas the O-glycans are predominantly core 1 and 2 type glycans with branched core 1 antennae (9). Arecent study demonstrated that CA125 from EOC patient sera has increased concentrations of core-fucosylated biantennary mono-sialylated glycans compared to that of controls and have decreased mostly bisecting biantennary and nonfucosylated glycans (15).

Lectins are the most widely used biorecognition agents for glycans and have been used to demonstrate glycosylation differences in soluble glycoproteins derived from malignant and benign tissues. For instance, fucosylation of a-fetoprotein (AFP), which reacts with the lectins Lens culinaris agglutinin and Aluria aurentia agglutinin, has been shown to discriminate AFP produced by liver cancer from that originating from nonmalignant liver (18-19). An assay using a 3-sulfated core1-specific galectin-4 lectin was recently reported to show superior clinical sensitivity and specificity over a conventional CA15-3 immunoassay for breast cancer (20). C-type lectin receptors (CLRs) present in cells of the immune system are glycan-binding receptors recognizing glycan structures in proteins and lipids. CLRs, such as macrophage galactose-type lectin (MGL) and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) showed increased binding to MUC1 and carcinoembryonic antigen of colon cancer tissue lysates, respectively, compared to the normal tissue from the same patients (21). Specific carbohydrate moieties 2-3- or 2-6-linked sialic acid and [beta]-galactosides are involved in the binding of CA125 to the lectins siglec-9 and galectin-1, respectively (22-23).

These results suggest that lectin assisted detection of tumor specific glycan structures on CA125 in serum samples is feasible, but the binding strength of the lectins, generally known to possess low affinities, must be improved for an analytically sensitive and robust clinical assay. Here, we demonstrate that differences in the glycosylation of CA125 from different origins can be efficiently detected using lectin-coated fluorescent lanthanide nanoparticle (NP) tracers. A panel of lectins (n = 16) including plant as well as 2 human lectins were tested, resulting in an assay that allowed preferential detection of ovarian cancer cell--derived CA125, and also improved discrimination between patients with EOC and endometriosis.

Materials and Methods

CLINICAL SAMPLES

CA125 samples used for glycoprofiling experiments were of 3 different origins: (a) pooled ascites fluid from 3 patients with liver cirrhosis (LC), (b) 5 homogenates of full-term placentas (Pla), and (c) CA125 from the OVCAR-3 ovarian cancer cell line (OvCa). CA125 from LC and Pla were used as normal/nonmalignant sources of the antigen to indicate the degree of unwanted cross-reactivity in the lectin assisted assays. Prospectively collected serum samples from patients with high-grade serous EOC (n = 21) or endometriosis (n = 121) as well as healthy controls (operated for tubal sterilization; n = 51) were used to analyze preoperative CA125 concentrations. Patient characteristics are presented in Table 1 (24). For differential EOC diagnostics, the most challenging group consisted of patients with marginally increased CA125 and abnormal ovarian findings in ultrasound. To simulate this situation, we selected serum samples with CA125 concentrations of 35-200 U/mL taken from 38 high-grade serous EOC in different phases of treatment and 44 endometriosis serum samples with a similar CA125 range. In addition, we measured longitudinal serum samples of 27 women with high-grade serous EOC during primary treatment and follow-up until disease progression. All EOC patients were treated for disseminated disease with surgery and platinum-based chemotherapy and had regular follow-up examinations to detect early recurrence. Patients had a clinically significant initial treatment response as judged by reduction of CA125 values but experienced disease progression, which was verified with radiologic and serologic parameters. The Ethics Committees of the Hospital District of Southwest Finland and University of Turku, Turku, Finland approved the use of clinical materials applied (ClinicalTrials.gov identifier NCT01276574 for EOC and NCT01301885 for endometriosis and healthy controls).

The CA125 isolated from the OVCAR-3 ovarian carcinoma cell line (OvCa-CA125), and 3 monoclonal anti-CA125 antibodies (MAbs) that specifically recognized different protein epitopes of CA125 (Ov185, Ov197, and OvK95) were kindly provided by Fujirebio Diagnostics (Goteborg, Sweden). Yellow streptavidin-coated low-fluorescence microtitration plates, wash buffer, and assay buffer were from Kaivogen Oy. Europium (IlI)-chelate--doped Fluora-Max[TM] polystyrene NPs (97 nm in diameter, [Eu.sup.+3]-NP) were purchased from Seradyn.

The lectins tested and their glycan-binding specificities are shown in Table 2. The plant lectins were obtained from Vector laboratories. The human recombinant CLRs proteins consisted of the extracellular region (carbohydrate recognition domain) fused with a human IgG1 Fc tail. The MGL-Fc and DC-SIGN-Fc were produced by Chinese hamster ovary cells as previously described (25) and obtained from the Department of Molecular Cell Biology and Immunology, VU University Medical Center (Amsterdam, The Netherlands).

PREPARATION OF NP-LECTIN BIOCONJUGATES

Amino groups of lectins were covalently coupled to activated carboxyl groups of the [Eu.sup.+3]-NPs using a previously described procedure (26) with some minor modifications. Activation was performed using N-hydroxysulfosuccinimide (sulfo-NHS)- and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC)-chemistries. Sulfo-NHS and EDC (Sigma) were incubated with [1e10.sup.12] NPs in 10 mmol/L phosphate buffer (pH 7.0) at final concentrations of 10 and 0.75 mmol/L, respectively at room temperature for 15 min. The concentration of lectins in the coupling reactions was 0.625 g/L. The coupling reactions were incubated for 2 h at room temperature under continuous mixing. Final washes and blocking of the remaining active groups were performed in Tris-based buffer (10 mmol/L Tris, 0.5 g/L NaN3, pH 8.5), and the NPs-lectin conjugates were stored in the same buffer supplemented with 2 g/L BSA at 4 [degrees]C. Before the first use, the particles were mixed thoroughly, sonicated, and centrifuged lightly (350 g, 5 min) to separate noncolloidal aggregates from the monodisperse suspension.

LABELING OF ANTIBODIES AND LECTINS WITH LANTHANIDE CHELATE

The Ov185 MAb and a panel of lectins were labeled with intrinsically fluorescent-[Eu.sup.+3]-chelate as described previously (27). Overnight labeling was carried out with 40-fold molar excess of chelate in 50 mmol/L carbonate buffer (pH 9.8) in a total volume of 500 pL at 4[degrees]C. The labeled antibody and lectins were separated from the unconjugated chelate by gel filtration using NAP[Tm]-5 and NAP-10 gel-filtration columns (GE Healthcare Life Sciences/Amersham Biosciences AB). The [Eu.sup.+3]-labeled MAb and lectins were stabilized with 1 g/L BSA (Bioreba) and stored at 4[degrees]C.

BIOTINYLATION OF ANTIBODIES AND PREPARATION OF SOLID-PHASE SURFACES

All 3 solid-phase antibodies were biotinylated for 4 h at room temperature, using a procedure described earlier (28). Biotinylated antibodies were purified with NAP-5 and NAP-10 gel-filtration columns using 50 mmol/L Tris-HCl (pH 7.75), containing 150 mmol/L NaCl and 0.5 g/L Na[N.sub.3]. The labeled antibodies were stored in 1 g/L BSA at 4[degrees]C.

IN-HOUSE TIME RESOLVED FLUORIMETRY IMMUNOASSAY FOR THE CA125 MEASUREMENT, AND CA125 NP-LECTINSSAY

The principle of the assay is schematically shown in Fig. 1. Biotinylated capture Ov185 or bio-Ov197 MAb (200 ng/ /well) were immobilized to streptavidin-coated microtiter wells in 100 [micro]L of Kaivogen assay buffer for 60 min at room temperature with shaking. After washing, 50 [micro]L of diluted standard/serum/plasma sample (1:5 in buffer solution) was added to each well and incubated for 60 min at room temperature with shaking. The captured CA125 antigen (from different origin) was detected using 3 different time-resolved fluorometry (TRF) assay formats. The [Eu.sup.+3]-chelate labeled Ov185 MAb detected the CA125 protein epitope whereas the [Eu.sup.+3]-chelate-labeled lectins and lectin-NP conjugates were used for detection of glycan epitopes.

For TRF CA125 immunoassay and CA125 lectin assay, 200 [micro]L Kaivogen assay buffer containing 25 ng of [Eu.sup.+3]-chelate labeled Ov185 MAb or lectin was added to each well for 1h at room temperature with shaking. TRF for europium was measured ([lambda]ex: 340 nm; [lambda]em: 615 nm) using Victor[TM] 1420 Multilabel counter (Perkin-Elmer Life Sciences), and after adding enhancement solution and incubating for 10 min with shaking the TRF signal was measured.

For the TRF CA125 lectin-NPs assay, 100 [micro]L assay buffer (with an additional 6 mmol/L Ca[Cl.sub.2]) containing 1 x [10.sup.7] Eu-NPs coated with lectin was added to each well for 2 h at room temperature with shaking. After incubation, the wells were washed 6 times with wash buffer. Then, TRF for [Eu.sup.+3] was measured ([[lambda].sub.ex]: 340 nm; [[lambda].sub.em]: 615 nm) from dry wells using Victor 1420 Multilabel counter using a modified standard europium protocol: measurement height 5 mm.

CA125 from different origins (Pla, LC, and OvCa) at 5, 50, and 100 U/mL concentrations in Tris-buffered saline with azide (50 mmol/L Tris-HCl, pH7.75, 9 g/L NaCl and 0.5 g/L NaN3) containing 75 g/L BSA (Sigma), were captured on 96-well plates using bio-Ov185 MAb and subsequently traced with individual lectins-NPs. Background subtracted specific signals and signal-to-background were recorded to determine which lectins best discriminated the different origins of CA125. Concentrations of CA125 were analyzed in serum samples by the ECLIA method (Modular E170 automatic analyzer, Roche Diagnostics) or ELISA analysis (Fujirebio Diagnostics) according to the manufacturer's instructions.

DATA ANALYSIS

Marker concentrations in disease groups were compared using Kruskal-Wallis one-way ANOVA with post hoc Dunn test on rank-transformed data. Statistical analyses were performed using SigmaStat software (Systat Software). Statistical difference was considered significant if the P value was <0.05.

Results

The concentrations of LC- and Pla-CA125 as quantified by the in-house CA125 immunoassay were 1358 and 2655 U/mL, respectively, using the CA125 from the OvCa as a standard. Consequently, the LC-, Pla- and OvCa-CA125 antigens within the concentration range of 5-2000 U/mL had close to identical potencies with the in-house sandwich immunoassay (see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol62/issue10).

CA125 LECTIN-[Eu.sup.+3]-CHELATE ASSAYS

Sixteen lectins with different glycan-binding specificities were used to explore and compare the glycosylation patterns of the 3 CA125 preparations. In the first experiment, CA125 was captured on microtiter wells by an anti-CA125 antibody and traced by the soluble lectins directly labeled with [Eu.sup.+3]-chelate. In summary, highly variable backgrounds and low specific signals were observed with the different lectins and no significant discrimination of CA125 from the 3 sources could be observed (data not shown).

CA125 NP-LECTIN ASSAYS

With the use of the 16 lectins coated on Eu-NP tracers, we observed different binding patterns to the antibody captured CA125 antigens (Fig. 2). The lectin-NPs tracers coated with Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin, Phaseolus vulgaris agglutinin-erythroagglutinin, soybean agglutinin, Sambucus nigra agglutinin, peanut aggluttin, Vicia villosa lectin (VVL), Aleuria aurantia lectin, Trichosanthes japonica agglutinin and Helix pomatia agglutinin exhibited strong preference for the Pla-CA125 antigen. Wheat germ agglutinin, Ricinus communis agglutinin and Ulex europaeus agglutinin-based NPs allowed detection of CA125 from all 3 different origins, albeit with variable efficiencies. Wheat germ agglutinin yielded a 3-fold higher signal with LC-CA125 compared with those observed in OvCa- and Pla-CA125, while Ricinus communis agglutinin detected both LC- and OvCa-CA125 2-fold better than Pla-CA125. Ulex europaeus agglutinin NPs in turn detected CA125 from all 3 origins equally. Six lectin NPs yielded a signal-to-background ratio close to 2 or higher when 5 U/mL of OvCa-CA125 was used. Of those, the MGL-based NPs tracer clearly discriminated CA125 from the OvCa from the 2 noncancerous preparations. The recombinant human C-type lectin MGL assay (CA125MGL) yielded more than 10-fold higher specific signals with OvCa-CA125 compared to Pla-CA125, and no signal was obtained with LC-CA125. The [CA125.sup.MGL] assay also showed the lowest background among the lectin-NPs-based assays tested, enabling a superior signal-to-background ratio with the OvCa-CA125 standard.

The analytical recovery of OvCa-CA125 standard from spiked plasma samples was 93%-109%. We compared the performance of the 3 anti-CA125 MAbs as capture antibodies in the [CA125.sup.MGL]assay. All 3 antibodies allowed efficient and highly similar discrimination of OvCa-CA125 from Pla-CA125 and LC-CA125. Ov185 MAb, the antibody also used in the initial experiments, showed the best performance due to the reproducibly low background signal. No hook effect was observed up to 2000 U/mL of OvCa-CA125 (Fig. 3A). The dose response curve and the precision profile of the [CA125.sup.MGL] assay using an OvCa-CA125 range from 0.1-1000 U/mL is shown in Fig. 3B. The analytical sensitivity, defined as the concentration of OvCa-CA125 required to give a signal equal to the mean of zero calibrator (n = 20) +3 times the SD, was 0.17 U/mL. A linear response was maintained up to 100 U/mL.

SERUM [CA125.sup.MGL] CONCENTRATIONS IN PATIENTS WITH EOC AND ENDOMETRIOSIS

We next applied the [CA125.sup.MGL] assay to the analysis of serum samples from healthy individuals, patients with endometriosis, and patients with high-grade serous EOC, and compared them with the conventional CA125 immunoassay values. The median preoperative CA125 values in serum samples of healthy controls, patients with endometriosis and patients with EOC were 6.4, 24.9 and 700 U/mL, respectively (Fig. 4A), showing significantly increased concentrations both in endometriosis and EOC as compared with healthy controls. In contrast, median preoperative [CA125.sup.MGL] values (Fig. 4B) did not significantly differ between healthy controls (0.486 U/mL) and patients with endometriosis (0.841 U/mL; P = 0.073). In the EOC group the median [CA125.sup.MGL] was 37.93 U/mL and was significantly higher than in endometriosis. We further studied serum samples from endometriosis and patients with EOC with marginally increased CA125 values (35-200 U/mL) to evaluate the discrimination between malignant and benign conditions by conventional CA125 immunoassay and the [CA125.sup.MGL] assay (Fig. 4, C and D). The [CA125.sup.MGL] assay provided significantly higher (6.1-fold, P <0.001) concentrations in the EOC serum samples compared with those from the patients with endometriosis (Fig. 4D). In addition, the difference between healthy controls and patients with endometriosis was reduced from 8.3-fold (P < 0.001) in the conventional assay (Fig. 4C) to 2.5-fold difference, still reaching significance (P = 0.005), with the [CA125.sup.MGL] assay.

[CA125.sup.MGL] IN EOC DISEASE PROGRESSION

We also measured [CA125.sup.MGL] concentrations in sequential serum samples of27 patients with high-grade serous EOC to evaluate the assay's ability to detect disease recurrence. All patients were treated with surgery and chemotherapy, and were under surveillance for disease progression. All patients had a significant initial treatment response as judged by both reduction of CA125 values and radiologic response criteria, but experienced a verified disease progression during follow-up (range 0-30.6 months after chemotherapy; see online Supplemental Table 1). Twenty-two out of 27 patients were disease-free during or after chemotherapy based on CA125 below the cutoff value (35 U/mL), 4 had lowest CA125 values between 35 and 46 U/mL, and 1 had 111U/mL. The median concentration of [CA125.sup.MGL] in samples obtained at or just before EOC progression was 7.97 U/mL.

The relative [CA125.sup.MGL]-values showed earlier increase in 37.0% (10/27, Fig. 5A) of patients with EOC with progressive disease and higher fold-increase in 29.6% (9/27, Fig. 5B) of the patients, suggesting better detection of disease progression in two thirds of the patients (Table 3). For 22.2% (6/27) of patients the 2 measurements did not markedly differ from another (Fig. 5C) while in 3 cases (11.1%) CA125 immunoassay was a clinically more sensitive indicator of disease progression (Fig. 5D).

Discussion

Here, we report the development of an assay for the analytically sensitive and quantitative detection of aberrant glycosylation on EOC-CA125, which provided improved preference for the cancer-associated isoforms of the antigen. We used a MAb detecting the protein epitope for the initial capture, and tested a panel of lectins for their ability to detect antibody captured CA125. In contrast to the poor analytical sensitivities and discriminating capabilities obtained with [Eu.sup.+3]-chelate--labeled lectins, lectin-coated [Eu.sup.+3]NPs generally provided highly improved signal-to-background ratios disclosing variable reactivity towards CA125 preparations from different sources. The highly improved analytical performance of the method applying lectin NPs is due to the signal amplification by the 30000 [Eu.sup.+3]-chelates packed within the 107 nm NPs, and of the avidity effect created by the high density of immobilized lectins on the particle. The [Eu.sup.+3]-chelate--doped NPs assisted lectin technology thus enabled the construction of a simple rapid two-step protocol suitable for sensitive glycan profiling of CA125 from different sources.

The combined specificities of the 16 lectins tested cover a range of commonly found human glycans. The obtained binding patterns illustrate the existence of subtle differences in glycosylation between the 3 CA125 preparations studied. Using an antibody capture based microarray for the analysis of serum CA125, Chen et al. (16) showed that GalNAc a (Tn) specific MAbs and lectins such as VVL can be used to measure GalNAc a (Tn) structures in CA125 that are present in EOC, while in our study VVL bound poorly only to Pla-CA125. The discrepancy between the results is not known. However, the technologies used differ, unlike Chen et al. we used TRF with lectin NPs for detection that does not require additional sialidase treatment (removal of the terminal sialic acid).

Of the numerous lectins investigated in our study, merely recombinant human MGL, and only when coated on NPs, showed good potential for preferential detection of OvCa-CA125 glycoforms over the 2 noncancerous CA125 preparations. MGL is a member of the type II family of CLRs and is expressed on human antigen presenting cells (29). Only CLR exclusively recognizes terminal A-acetylgalactosamine (GalNAc) residues, including sialylated and nonsialylated Tn antigen and LacdiNAc, a well-known human carcinoma-associated epitope (35). In our study, the Tn antigen recognizing lectins, Helixpomatia agglutinin and VVL did not bind to OvCa-CA125, indicating that in OvCa-MGL was not binding to Tn-antigen but likely to sialyl Tn antigen or the LacdiNAc. Binding of human recombinant MGL lectin with formalin-fixed, paraffin-embedded cancerous mammary tissues (31) and with captured MUC1 from colon cancer tissue lysate (21) have been reported. A recent study by Lenos et al. demonstrated that MGL binding to the lesions correlated with poor survival in colon cancer patients (32). To the best of our knowledge, this is the first study to report MGL's specificity for OvCa-CA125.

Apart from the unique binding specificity of the human MGL lectin itself, the use of the [Eu.sup.+3]-NPs in combination with a CA125 MAb for initial capture constitutes the central technical concept behind the novel [CA125.sup.MGL] assay with an analytical specificity that strongly prefers the cancer-associated glycan epitopes. Based on the published literature describing the nature of CA125 glycosylation in cancer and noncancerous states, the MGL preference for cancer CA125 could not have been predicted. The very low background of the [CA125.sup.MGL] assay is a substantial advantage in reaching the very low detection of the assay, while excessive background signals were observed by several of the other lectins tested. It is also conceivable that the MGL-reactive glycan epitope can be present at several positions on CA125, being a large 200-2000 kDa glycoprotein. This provides a possibility for multiple high avidity binding sites for MGL NPs even at low CA125 concentration. The excellent linearity of the response over the whole assay range (0.1-100 U/L) is in agreement with this possibility. Since the MGL assay is expected to recognize yet uncharacterized CA125 isoforms, the proportion of which in the OvCa-CA125 is also not known, the calibration of the assay cannot be firmly established at this stage. Thus, one of the future tasks is to characterize in detail the CA125 form(s) recognized by the MGL coated NPs.

From a clinical point of view, there is an urgent need for assays with improved clinical specificity and sensitivity for ovarian lesions. In the diagnostic routine, the marginally increased CA125 values constitute a common problem in the discrimination between EOC and benign conditions, especially endometriosis, and can cause unnecessary interventions and costs. The novel [CA125.sup.MGL] assay reduces the CA125 reactivity in serum samples of endometriosis patients and thereby helps to identify patients with malignant conditions. The combination of the conventional CA125 assay together with the [CA125.sup.MGL] assay may provide further means to differentiate endometriosis from EOC, and could improve the diagnosis of endometriosis patients as well. The [CA125.sup.MGL] assay also enabled earlier detection of disease progression compared to the conventional CA125 assay, potentially enabling earlier therapeutic interventions. Based on these findings, we hypothesize that the [CA125.sup.MGL] assay may have the potential for early detection of EOC. Because the patient samples included in this proof-of-principle technical report are relatively limited, further validation studies are called upon to establish the clinical performance of [CA125.sup.MGL] assay for primary diagnosis of EOC, and monitoring the disease relapse, and progression and therapeutic responses.

Finally, using appropriate combinations of lectins and antibodies, applying the test concept presented here can also be explored for other diagnostic targets, where changes in glycosylation are indicative of an ongoing disease process.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met thefollowing 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: K. Gidwani, Department of Biotechnology (DBT), Government of India; U. Lamminmaki, PROVATECT FINLAND funded by TEKES (decision number 40089/14); O. Carpen, Tekes funding.

Expert Testimony: None declared.

Patents: K. Gidwani, Patent application no. FI20155531; K. Pettersson,

Finnish patent application no. FI20155531.

Role of Sponsor: No sponsor was declared.

Acknowledgments: We gratefully acknowledge Henri Lahteenmaki and Jenna Jacobino at the Department of Biotechnology, University of Turku, for excellent technical assistance.

References

(1.) Goonewardene TI, Hall MR, Rustin GJ. Management of asymptomatic patients on follow-up for ovarian cancer with rising CA-125 concentrations. Lancet Oncol 2007; 8:813-21.

(2.) Scholler N, Urban N. CA125 in ovarian cancer. Biomark Med 2007; 1:513-23.

(3.) Mai PL, Wentzensen N, Greene MH. Challenges related to developing serum-based biomarkers for early ovarian cancer detection. Cancer Prev Res (Phila) 2011; 4: 303-6.

(4.) Jacobs IJ, Menon U, Ryan A, Maharaj A G, Burnell M, Jatinderpal K, et al. Ovarian cancer screening and mortality in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS): a randomised controlled trial. Lancet 2016; 387:945-56.

(5.) Hakomori S. Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Cancer Res 1996; 56:5309-18.

(6.) Fuster MM, Esko JD. The sweet and sour of cancer: glycans as novel therapeutic targets. Nat Rev Cancer 2005; 5:526-42.

(7.) Satomaa T, Heiskanen A, Leonardsson I, Angstrom J, Olonen A, Blomqvist M, et al. Analysis of the human cancer glycome identifies a novel group of tumor-associated W-acetylglucosamine glycan antigens. Cancer Res 2009; 69:5811-9.

(8.) Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci USA 2002; 99:10231-3.

(9.) Kui WN, Easton RL, Panico M, Sutton-Smith M, Morrison JC, Lattanzio FA, et al. Characterization of the oligosaccharides associated with the human ovarian tumor marker CA125. J Biol Chem 2003; 278:28619-34.

(10.) Saldova R, Wormald MR, Dwek RA, Rudd PM. Glycosylation changes on serum glycoproteins in ovarian cancer may contribute to disease pathogenesis. Dis Markers 2008; 25:219-32.

(11.) Garcia E, Andrews C, Hua J, Kim HL, Sukumaran DK, Szyperski T, Odunsi K. Diagnosis of early stage ovarian cancer by 1H NMR metabonomics of serum explored by use of a microflow NMR probe. J Proteome Res 2011; 10:1765-71.

(12.) Kyselova Z, Mechref Y, Al Bataineh MM, Dobrolecki LE, Hickey RJ, Vinson J, et al. Alterations in the serum glycome due to metastatic prostate cancer. J Proteome Res 2007; 6:1822-32.

(13.) Kirmiz C, Li B, An HJ, Clowers BH, Chew HK, Lam KS, et al. A serum glycomics approach to breast cancer biomarkers. Mol Cell Proteomics 2007; 6:43-55.

(14.) Isailovic D, Kurulugama RT, Plasencia MD, Stokes ST, Kyselova Z, Goldman R, et al. Profiling of human serum glycans associated with liver cancer and cirrhosis by IMS-MS. J Proteome Res 2008; 7:1109-17.

(15.) Saldova R, Struwe WB, Wynne K, Elia G, Duffy MJ, Rudd PM. Exploring the glycosylation of serum CA125. Int J Mol Sci 2013; 14:15636-54.

(16.) Chen K, Gentry-Maharaj A, Burnell M, Steentoft C, Marcos-Silva L, Mandel U, et al. Microarray glycoprofiling of CA125 improves differential diagnosis of ovarian cancer. J Proteome Res 2013; 12:1408-18.

(17.) Yin BW, Lloyd KO. Molecular cloning of the CA125 ovarian cancer antigen: identification as a new mucin, MUC16. J Biol Chem 2001; 276:27371-5.

(18.) Taketa K, EndoY, Sekiya C, Tanikawa K, Koji T, Taga H, et al. A collaborative study for the evaluation of lectin-reactive alpha-fetoproteins in early detection of hepatocellular carcinoma. Cancer Res 1993; 53:5419-23.

(19.) Korekane H, Hasegawa T, Matsumoto A, Kinoshita N, Miyoshi E, Taniguchi N. Development of an antibody-lectin enzyme immunoassay for fucosylated alpha-fetoprotein. Biochim Biophys Acta 2012; 1820:1405-11.

(20.) Ideo H, Hinoda Y, Sakai K, Hoshi I, Yamamoto S, Oka M, et al. Expression of mucin 1 possessing a 3'-sulfated core1 in recurrent and metastatic breast cancer. Int J Cancer 2015; 137:1652-60.

(21.) Saeland E, Belo AI, MongeraS, van D, I, Meijer GA, van KY. Differential glycosylation of MUC1 and CEACAM5 between normal mucosa and tumour tissue of colon cancer patients. Int J Cancer 2012; 131:117-28.

(22.) Belisle JA, Horibata S, Jennifer GA, Petrie S, Kapur A, Andre S, et al. Identification of Siglec-9 as the receptor for MUC16 on human NK cells, B cells, and monocytes. Mol Cancer 2010; 9:118.

(23.) Seelenmeyer C, Wegehingel S, Lechner J, Nickel W. The cancer antigen CA125 represents a novel counter receptor for galectin-1. J Cell Sci 2003; 116:1305-18.

(24.) Mutch DG, Prat J. 2014 FIGO staging for ovarian, fallopian tube and peritoneal cancer. Gynecol Oncol 2014; 133:401-4.

(25.) van Vliet SJ, van LE, Saeland E, Aarnoudse CA, Appelmelk B, Irimura T, et al. Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells. Int Immunol 2005; 17:661-9.

(26.) Soukka T, Harma H, Paukkunen J, Lovgren T. Utilization of kinetically enhanced monovalent binding affinity by immunoassays based on multivalent nanoparticle-antibody bioconjugates. Anal Chem 2001; 73:2254-60.

(27.) von LP, Rosenberg J, Pettersson K, Takalo H. A europium chelate for quantitative point-of-care immunoassays using direct surface measurement. Anal Chem 2003; 75:3193-201.

(28.) Eriksson S, Halenius H, Pulkki K, Hellman J, Pettersson K. Negative interference in cardiactroponin I immunoassays by circulating troponin autoantibodies. Clin Chem 2005; 51:839-47.

(29.) Singh SK, Streng-Ouwehand I, Litjens M, Weelij DR, Garcia-Vallejo JJ, van Vliet SJ, et al. Characterization of murine MGL1 and MGL2 C-type lectins: distinct glycan specificities and tumor binding properties. Mol Immunol 2009; 46:1240-9.

(30.) van KY, Ilarregui JM, van Vliet SJ. Novel insights into the immunomodulatory role of the dendritic cell and macrophage-expressed C-type lectin MGL. Immunobiology 2015; 220:185-92.

(31.) Nollau P, Wolters-Eisfeld G, Mortezai N, Kurze AK, Klampe B, Debus A, et al. Protein domain histochemistry (PDH): binding of the carbohydrate recognition domain (CRD) of recombinant human glycoreceptor CLEC10A (CD301) to formalin-fixed, paraffin-embedded breast cancer tissues. J Histochem Cytochem 2013; 61:199-205.

(32.) Lenos K, Goos JA, Vuist IM, den Uil SH, Delis-van Diemen PM, Belt EJ, et al. MGL ligand expression is correlated to BRAF mutation and associated with poor survival of stage III colon cancer patients. Oncotarget 2015; 6:26278-90.

(33.) Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril 1997; 67:817-21.

Kamlesh Gidwani, [1] * ([dagger]) Kaisa Huhtinen, [2] ([dagger]) Henna Kekki, [1] Sandra van Vliet, [3] Johanna Hynninen, [4] Niina Koivuviita, [5] Antti Perheentupa, [4] Matti Poutanen, [6] Annika Auranen, [4, 7] Seija Grenman, [4] Urpo Lamminmaki, [1] Olli Carpen, [2, 8] Yvette van Kooyk, [3] and Kim Pettersson [1]

[1] Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland; [2] Department of Pathology, Medicity research laboratories, University of Turku and Turku University Hospital, Turku, Finland; [3] Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands; [4] Department of Obstetrics and Gynecology, University of Turku and Turku University Hospital, Turku, Finland; [5] Department of Medicine, University of Turku and Turku University Hospital, Turku, Finland; [6] Department of Physiology, Institute of Biomedicine, and Turku Center for Disease Modeling, University of Turku, Finland; [7] Department of Obstetrics and Gynecology, Tampere University Hospital, Tampere, Finland; [8] Department of Pathology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

* Address correspondence to this author at: BioCity 6th Floor, Rm. 6079, Molecular Biotechnology and Diagnostics, Department of Biochemistry, Tykistokatu 6A 6th floor, University of Turku, FI-20014 Yliopistoy, Finland. Fax +358-2-333-8050; e-mail kamgid@utu.fi.

([dagger]) Kamlesh Gidwani and Kaisa Huhtinen contributed equally to the work, and both should be considered as first authors.

Received April 4, 2016; accepted June 23, 2016.

Previously published online at DOI: 10.1373/clinchem.2016.257691

[9] Nonstandard abbreviations: CA125, serum cancer antigen 125; EOC, epithelial ovarian cancer; AFP, [alpha]-fetoprotein; CLRs, C-type lectin receptors; MGL, macrophage galactose-type lectin; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin; NP, nanoparticle; LC, liver cirrhosis; Pla, full-term placentas; OvCa, OVCAR-3 ovarian cancer cell line; MAb, monoclonal antibodies; TRF, time-resolved fluorimetry; OvCa-CA125, CA125 purified from OvCa; Pla-CA125, CA125 from placental extract; LC-CA125, CA125 in ascetic fluid of liver cirrhosis.

Caption: Fig. 1. Schematic representation of the in-houseTRF CA125 assays. (A) Conventional CA125 immunoassay, in which the capture and tracer MAbs detect different protein epitopes of CA125, (B) A combined immuno-lectin assay, in which MAb-captured CA125 is traced with lectin-[Eu.sup.+3] chelate, which binds to glycan moieties of CA125. (C) A NP-based immuno-lectin assay, in which lectins are coated on the surface of [Eu.sup.+3]-NPs.

Caption: Fig. 2. Lectin NPs binding to CA125 from (A) primary OvCa cell line OVCAR-3 (OvCa-CA125), (B) placental homogenate (Pla-CA125), and (C) liver cirrhosis-derived ascites fluid (LC-CA125). The x axis shows different lectin NPs used while they axis shows the signal to background ratios. PHA-E, Phaseolus vulgaris agglutinin-erythroagglutinin; SBA, soybean agglutinin; WGA, wheat germ agglutinin; SNA, Sambucus nigra agglutinin; PNA, peanut agglutinin; WFA, Wisteria floribunda agglutinin; PSA, Pisum sativum agglutinin; VVL, Vicia villosa lectin; RCA, Ricinus communis agglutinin; AAL, Aleuria aurantia lectin; TJA, Trichosanthes japonica agglutinin; MAA, Maackia amurensis agglutinin II; UEA, Ulex europaeus agglutinin; HPA, Helix pomatia agglutinin.

Caption: Fig. 3. CA125MGL NPs-lectin immunoassay: (A) OvCa-CA125 resulted in markedly highersignal to background ratio as compared to that observed for CA125 from benign origin.

The error bars represent SD measured from 3 replicates of 3 different days. (B) Calibration curve (solid line) and precision profiles (dashed line) of [CA125.sup.MGL] assay for OvCa-CA125.

Caption: Fig. 4. Discrimination of EOC from benign endometriosis and healthy controls using CA125 immunoassay (A, C) and [CA125.sup.MGL] assay (B, D). (A) CA125 in preoperative high-grade serous EOC(n = 21) and endometriosis (n = 121) were significantly higher than in healthy controls (n = 51) with conventional CA125 immunoassay (P < 0.001). (B), No significant difference between endometriosis and healthy controls was observed in [CA125.sup.MGL] concentrations while preoperative EOC concentrations were significantly higher (P < 0.001). (C), EOC (n = 38) and endometriosis (n =44) samples with marginally increased CA125 concentrations (35-200 U/mL), which are clinically the most challenging for diagnostics, did not differ with CA125 immunoassay, while (D) [CA125.sup.MGL] concentrations remained significantly different.

Caption: Fig. 5. Relative serum CA125 concentrations during early EOC progression after response to primary treatment measured with CA125 immunoassay and [CA125.sup.MGL]-assay. (A) [CA125.sup.MGL] assay showed earlier increase in 37.0% of EOC progression (example cases M014, M054, M062) and (B) higher fold-increase in 29.6% of the cases (example cases M017, M025, M042).

For 22.2% of cases the 2 measurements did not markedly differ from another (C; example case M012) while in 3 cases (11.1%) CA125 immunoassay was a more sensitive indicator of disease progression (D; example case M032). Relative concentrations were calculated by dividing measured concentration by a cutoff value of each marker i.e., 35 U/mL for CA125 immunoassay and 2 U/mLfor [CA125.sup.MGL] NP-lectin assay. IA, immunoassay.
Table 1. Patient characteristics.

                                                         Preoperative

Healthy         Number of patients   --                       51
                Age at diagnosis,    Median (range)        38(32-48)
                years

Endometriosis   Number of patients                            121
                Disease stage (a)    1                     16(13.2%)
                                     2                     15(12.4%)
                                     3                    33 (-27.3%)
                                     4                     57(47.1%)
                Ovarian                                   64 (52.9%)
                endometrioma
                Age at diagnosis,    Median (range)       31 (19-48)
                years

Ovarian         Number of patients                            21
cancer          Age at diagnosis,    Median (range)       70 (57-79)
                years
                Histology            High grade serous     21 (100%)
                CA125, U/mL          Median (range)      700 (55-2940)
                Stage (FIGO          IC2
                2014) (b)            IIB                   1 (4.8%)
                                     IIIB                  1 (4.8%)
                                     IIIC                  9 (42.9%)
                                     IVA                   4(19.0%)
                                     IVB                   6 (28.6%)
                Treatment line       PDS                   8(38.1%)
                                     NACT                  13(61.9%)
                Residual disease     No macroscopic        2 (9.5%)
                                       disease
                                     1-10 mm              11 (52.4%)
                                     >10 mm                6 (28.6%)
                                     Unknown               2 (9.5%)
                Primary therapy      Complete              9 (42.9%)
                outcome              response
                                     Partial response      6 (28.6%)
                                     Stable disease        1 (4.8%)
                                     Progressive           5 (23.8%)
                                     disease

                                                         Marginally
                                                          increased
                                                            CA125
Healthy         Number of patients   --
                Age at diagnosis,    Median (range)
                years

Endometriosis   Number of patients                           44
                Disease stage (a)    1                    1 (2.3%)
                                     2                    3 (6.8%)
                                     3                    10(22.7%)
                                     4                   30 (68.2%)
                Ovarian                                  29 (65.9%)
                endometrioma
                Age at diagnosis,    Median (range)      31 (19-46)
                years

Ovarian         Number of patients                           38
cancer          Age at diagnosis,    Median (range)      65 (55-80)
                years
                Histology            High grade serous    38(100%)
                CA125, U/mL          Median (range)      70 (41-198)
                Stage (FIGO          IC2                  1 (2.6%)
                2014) (b)            IIB                  1 (2.6%)
                                     IIIB                 1 (2.6%)
                                     IIIC                 19 (50%)
                                     IVA                  5(13.2%)
                                     IVB                 11 (28.9%)
                Treatment line       PDS                 12 (31.6%)
                                     NACT                26 (68.4%)
                Residual disease     No macroscopic       8(21.1%)
                                       disease
                                     1-10 mm             22 (57.9%)
                                     >10 mm               6(15.8%)
                                     Unknown              2 (5.3%)
                Primary therapy      Complete             19(50.0%)
                outcome              response
                                     Partial response     10(26.3%)
                                     Stable disease       1 (2.6%)
                                     Progressive          8(21.1%)
                                     disease

                                                           Disease
                                                         progression

Healthy         Number of patients   --
                Age at diagnosis,    Median (range)
                years

Endometriosis   Number of patients
                Disease stage (a)    1
                                     2
                                     3
                                     4
                Ovarian
                endometrioma
                Age at diagnosis,    Median (range)
                years

Ovarian         Number of patients                            27
cancer          Age at diagnosis,    Median (range)       65 (37-79)
                years
                Histology            High grade serous     28(100%)
                CA125, U/mL          Median (range)      63 (14-462)
                Stage (FIGO          IC2
                2014) (b)            IIB
                                     IIIB                  1 (3.6%)
                                     IIIC                 12 (42.9%)
                                     IVA                  4 (14.3%)
                                     IVB                  11 (39.3%)
                Treatment line       PDS                   9(32.1%)
                                     NACT                 19(67.9%)
                Residual disease     No macroscopic        6(21.4%)
                                       disease
                                     1-10 mm              18(64.3%)
                                     >10 mm               4 (14.3%)
                                     Unknown
                Primary therapy      Complete             15(53.6%)
                outcome              response
                                     Partial response      9(32.1%)
                                     Stable disease
                                     Progressive          4 (14.3%)
                                     disease

(a) The revised American Society for Reproductive Medicine criteria
(33).

(b) The FIGO 2014 ovarian cancer staging guideline [Mutch and Prat
(24)] was used.

Table 2. Lectins used.

Lectin name                         Major carbohydrate binding
                                            specificity

Soybean agglutinin                Terminal [alpha]- or
                                    -[beta]-linked GalNAc
Sambucus nigra agglutinin         Sialic acid [alpha] (2-6) Gal
Peanut agglutinin                 Gal[beta]1-3 GalNAc (terminal)
Maackia amurensis agglutinin      [alpha]2-3-Linked sialic acids
  II
Aleuria aurantia lectin           [alpha]1-6Fuc
Ulex europaeus agglutinin         Fuc[alpha]1-2Gal
Phaseolus vulgaris agglutinin-    Bisecting GlcNAc
  erythroagglutinin
Ricinus communis agglutinin       Gal-[beta]1-4GlcNAc
Wheat germ agglutinin             Terminal N-acetylglucosamine
                                    or chitobiose
Wisteria floribunda agglutinin    GalNAc[alpha] or [beta]-3 or 6
                                    position of galactose
Pisum sativum agglutinin          [alpha]-Mannose
Vicia villosa lectin              Terminal [alpha]-or [beta]-
                                    linked GalNAc (Tn antigen)
Helix pomatia agglutinin          GalNAc (Tn antigen)
Trichosanthes japonica            Fuc [alpha] 1-2Gal and [beta]-GalNAc
  agglutinin
Macrophage galactose-type         Terminal [alpha]-or
  lectin                            -[beta]-linked GalNAc
Dendritic cell-specific           Nonsialylated Lewis antigens
  intercellular adhesion            and high-mannose type
  molecule-3-grabbing non-          structures
  integrin

Table 3. Ability of CA125-MGL and CA125-immunoassay (IA) to predict
EOC progression.

CA125-MGL vs CA125-IA             Count    %

Eearlier detection with CA125-    10/27   37.0
  MGL
Stronger increase in CA125-MGL    8/27    29.6
  in progression
CA125-MGL and CA125-IA are        6/27    22.2
  similar
Later or weaker detection with    3/27    11.1
  CA125-MGL
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Title Annotation:Cancer Diagnostics
Author:Gidwani, Kamlesh; Huhtinen, Kaisa; Kekki, Henna; van Vliet, Sandra; Hynninen, Johanna; Koivuviita, N
Publication:Clinical Chemistry
Date:Oct 1, 2016
Words:6374
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