Lectin nanoparticle assays for detecting breast cancer-associated glycovariants of cancer antigen 15-3 (CA15-3) in human plasma

Cancer antigen 15-3 (CA15-3) is widely utilized for monitoring metastatic breast cancer (BC). However, its utility for early detection of breast cancer is severely limited due to poor clinical sensitivity and specificity. The glycosylation of CA15-3 is known to be affected by BC, and therefore it might offer a way to construct CA15-3 glycovariant assays with improved cancer specificity. To this end, we performed lectin-based glycoprofiling of BC-associated CA15-3. CA15-3 expressed by a BC cell line was immobilized on microtitration wells using an anti-CA15-3 antibody. The glycosylation of the immobilized CA15-3 was then detected by using lectins coated onto europium (III)-doped nanoparticles (Eu+3-NPs) and measuring the time-resolved fluorescence of Eu. Out of multiple lectin-Eu+3-NP preparations, wheat germ agglutinin (WGA) and macrophage galactose-type lectin (MGL) -Eu3+-NPs bound to the BC cell line-dericed CA15-3 glycovariants (CA15-3Lectin). To evaluate the clinical performance of these two lectin-based assays, plasma samples from metastatic BC patients (n = 53) and healthy age-matched women (n = 20).Plasma CA15-3Lectin measurements better distinguished metastatic BC patients from healthy controls than the conventional CA15-3 immunoassay. At 90% specificity, the clinical sensitivity of the assays was 66.0, 67.9 and 81.1% for the conventional CA15-3, CA15-3MGL and CA15-3WGA assays, respectively. Baseline CA15-3MGL and CA15-3WGA were correlated to conventional baseline CA15-3 levels (r = 0.68, p0.001, respectively). However, very low baseline CA15-3MGL levels ≤ 5 U/mL were common in this metastatic breast cancer patient population.In conclusion, the new CA15-3Lectin concept could considerably improve the clinical sensitivity of BC detection compared to the conventional CA15-3 immunoassays and should be validated further on a larger series of subjects with different cancer subtypes and stages

Lectin nanoparticle assays for detecting breast cancer-associated glycovariants of cancer antigen 15-3 (CA15-3) in human plasma

Cancer antigen 15–3 (CA15-3) is widely utilized for monitoring metastatic breast cancer (BC). However, its utility for early detection of breast cancer is severely limited due to poor clinical sensitivity and specificity. The glycosylation of CA15-3 is known to be affected by BC, and therefore it might offer a way to construct CA15-3 glycovariant assays with improved cancer specificity. To this end, we performed lectin-based glycoprofiling of BC-associated CA15-3. CA15-3 expressed by a BC cell line was immobilized on microtitration wells using an anti-CA15-3 antibody. The glycosylation of the immobilized CA15-3 was then detected by using lectins coated onto europium (III)-doped nanoparticles (Eu+3-NPs) and measuring the time-resolved fluorescence of Eu. Out of multiple lectin-Eu+3-NP preparations, wheat germ agglutinin (WGA) and macrophage galactose-type lectin (MGL) -Eu3+-NPs bound to the BC cell line-dericed CA15-3 glycovariants (CA15-3Lectin). To evaluate the clinical performance of these two lectin-based assays, plasma samples from metastatic BC patients (n = 53) and healthy age-matched women (n = 20).Plasma CA15-3Lectin measurements better distinguished metastatic BC patients from healthy controls than the conventional CA15-3 immunoassay. At 90% specificity, the clinical sensitivity of the assays was 66.0, 67.9 and 81.1% for the conventional CA15-3, CA15-3MGL and CA15-3WGA assays, respectively. Baseline CA15-3MGL and CA15-3WGA were correlated to conventional baseline CA15-3 levels (r = 0.68, p0.001, respectively). However, very low baseline CA15-3MGL levels ≤ 5 U/mL were common in this metastatic breast cancer patient population.In conclusion, the new CA15-3Lectin concept could considerably improve the clinical sensitivity of BC detection compared to the conventional CA15-3 immunoassays and should be validated further on a larger series of subjects with different cancer subtypes and stages.</p

Diagnostic potential of nanoparticle aided assays for MUC16 and MUC1 glycovariants in ovarian cancer

Our study reports the discovery and evaluation of nanoparticle aided sensitive assays for glycovariants of MUC16 and MUC1 in a unique collection of paired ovarian cyst fluids and serum samples obtained at or prior to surgery for ovarian carcinoma suspicion. Selected glycovariants and the immunoassays for CA125, CA15-3 and HE4 were compared and validated in 347 cyst fluid and serum samples. Whereas CA125 and CA15-3 performed poorly in cyst fluid to separate carcinoma and controls, four glycovariants including MUC16(MGL), MUC16(STn), MUC1(STn) and MUC1(Tn) provided highly improved separations. In serum, the two STn glycovariants outperformed conventional CA125, CA15-3 and HE4 assays in all subcategories analyzed with main benefits obtained at high specificities and at postmenopausal and early-stage disease. Serum MUC16(STn) performed best at high specificity (90%-99%), but sensitivity was also improved by the other glycovariants and CA15-3. The highly improved specificity, excellent analytical sensitivity and robustness of the nanoparticle assisted glycovariant assays carry great promise for improved identification and early detection of ovarian carcinoma in routine differential diagnostics.Peer reviewe

Primary breast cancer biomarkers based on glycosylation and extracellular vesicles detected from human serum

Background Breast cancer is a very common cancer that can be severe if not discovered early. The current tools to detect breast cancer need improvement. Cancer has a universal tendency to affect glycosylation. The glycosylation of circulating extracellular vesicle-associated glycoproteins, and mucins may offer targets for detection methods and have been only explored in a limited capacity. Aim Our aim was to develop an approach to detect the aberrant glycosylation of mucins and extracellular vesicle-associated glycoproteins from human sera using fluorescent nanoparticles, and preliminarily evaluate this approach for the differential diagnosis of breast cancer. Methods and results The assay involved immobilizing glycosylated antigens using monoclonal antibodies and then probing their glycosylation by using lectins and glycan-specific antibodies coated on Eu+3-doped nanoparticles. Detection of mucin 1 and mucin 16 glycosylation with wheat germ agglutinin, and detection of the extracellular vesicle-associated CD63 were found to have better diagnostic ability for localized breast cancer than the conventional assays for mucin 1 and mucin 16 based tumor markers when the receiver operating characteristics were compared. Conclusions These results indicate that successful differential diagnosis of primary breast cancer may be aided by detecting cancer-associated glycosylation of mucin 1 and mucin 16, and total concentration of CD63, in human serum.</p

Diagnostic potential of nanoparticle aided assays for MUC16 and MUC1 glycovariants in ovarian cancer

Our study reports the discovery and evaluation of nanoparticle aided sensitive assays for glycovariants of MUC16 and MUC1 in a unique collection of paired ovarian cyst fluids and serum samples obtained at or prior to surgery for ovarian carcinoma suspicion. Selected glycovariants and the immunoassays for CA125, CA15-3 and HE4 were compared and validated in 347 cyst fluid and serum samples. Whereas CA125 and CA15-3 performed poorly in cyst fluid to separate carcinoma and controls, four glycovariants including MUC16(MGL), MUC16(STn), MUC1(STn) and MUC1(Tn) provided highly improved separations. In serum, the two STn glycovariants outperformed conventional CA125, CA15-3 and HE4 assays in all subcategories analyzed with main benefits obtained at high specificities and at postmenopausal and early-stage disease. Serum MUC16(STn) performed best at high specificity (90%-99%), but sensitivity was also improved by the other glycovariants and CA15-3. The highly improved specificity, excellent analytical sensitivity and robustness of the nanoparticle assisted glycovariant assays carry great promise for improved identification and early detection of ovarian carcinoma in routine differential diagnostics

Määritysmenetelmän kehittäminen proteiiniadsorption tutkimiseen nanopartikkeleista

Opinnäytetyö suoritettiin Turun yliopiston lääketieteellisen tiedekunnan Biofysiikan laboratoriossa. Työ edisti osaltaan laboratorion tavoitteita kehittää uusia menetelmiä biomolekyylien vuorovaikutusten tutkimiseen luminesenssiin pohjautuvin menetelmin. Kehitetyn menetelmän oli tarkoitus määrittää nanopartikkelien pinnoille tapahtuvan proteiiniadsorption täyttöastetta. Menetelmä perustui aikaerotteiseen fluoresenssiin ja resonanssienergian siirtoon (RET) europiumkelaattia sisältävien nanopartikkelien ja leimatun proteiinin välillä. Leimattu proteiini toimi RET-parin akseptorina ja kilpaili adsorboitumisesta näytepartikkeleihin adsorboituneen proteiinin kanssa. Leimatun proteiinin sitoutuminen näytepartikkeleihin heikensi RET-signaalia, joten RET-signaalin ja adsorption asteen välillä oli korrelaatio. Työssä verrattiin kehitettyä menetelmää kirjallisuudessa esitettyyn menetelmään, jossa näyteproteiini leimattiin europiumkelaatilla. Menetelmien havaittiin antavan tietoa proteiiniadsorption täyttöasteesta toisiaan vastaavilla tarkkuuksilla. Selvitettiin myös näytteen pitoisuuden ja näytepartikkelien koon vaikutusta määritykseen. Havaittiin, että näytemäärää säätelemällä voitiin paremmin havaita eroja tietyillä adsorption täyttöasteilla, sillä näytepartikkelien kokonaispinta-ala muuttui määrityksessä suhteessa näytteen pitoisuuteen. Näytemäärää lisäämällä voitaisiin siis havaita eroja paremmin esimerkiksi lähes saturoituneista partikkeleista. Proteiiniadsorption astetta voitiin mitata eri kokoisista ja hieman erilaisia pinnan ominaisuuksia omaavista partikkeleista. Yritettiin myös kehittää menetelmä, jossa näytepartikkelien pesuvaihe voitaisiin jättää pois ja mitata adsorpoitumatta jääneen proteiinin pitoisuuden laskua. Menetelmää ei vielä saatu toimimaan ilman pesuvaihetta, mutta tarkempi tutkimus voi osoittaa pesuvaiheen turhaksi. Kehitetyllä menetelmällä on potentiaalia nopeaksi ja tarkaksi proteiiniadsorption asteen mittausmenetelmäksi. Menetelmän käytettävyyttä rajoittaa vain leimatun proteiinin adsorboituvuus näytemateriaalin pinnalle.The thesis project was conducted in the Laboratory of Biophysics of the Faculty of Medicine, University of Turku. The study was performed to promote the recent efforts of the Laboratory to develop novel luminescence-based methods for investigating the interactions of biomolecules. The method was developed for the determination of the degree of protein adsorption onto nanoparticles. It was based on the time-resolved fluorescence and resonance energy transfer (RET) between nanoparticles doped with a europium chelate and a labeled protein. The labeled protein acted as the acceptor of the RET pair and competed in adsorption with the protein adsorbed onto the particles in the sample.. The adsorption of the labeled protein onto the sample decreased the RET signal. Thus, there was a correlation between the degree of adsorption and the RET signal. The developed method was compared to a literature method in which the sample protein was labeled with a europium chelate. Both methods were observed to give information on the degree of protein adsorption with similar accuracy. The effects of sample concentration and the size of sample particles on the method were also assessed. It was observed that the alteration of the sample concentrations enabled improved distinction of the degree of adsorption at certain surface coverages as the sample concentration is relative to the total surface area in the sample. Alteration of the sample concentration could thus provide more detailed information, for example, on the slight differences near saturation. The degree of protein adsorption was also found to be measurable from particles of different sizes and slightly different surface properties. A method was also investigated to measure the decrease in non-adsorbed protein due to adsorption leaving out the washing steps of samples. The results of this test were preliminary, but further studies may prove the sample washing step unnecessary. The developed method is potentially a fast and accurate assay for studying the degree of protein adsorption on an extensive range of sample materials. The range of sample materials is limited to those adsorbing the labeled protein

Mahdollisuudet karsinoembryonaalisen antigeenin glykaanirakenteiden hyödyntämiseen suolistosyöpädiagnostiikassa. Karsinoembryonaalisen antigeenin glykaanirakenteita tunnistava lektiininanopartikkelimääritys

Suolistosyöpä on vaikeasti havaittava ja yleinen syöpätauti, jonka syntyyn vaikuttavat geneettiset ja elämäntavoista johtuvat riskitekijät. Suolistosyövän havaitsemiseen ja hoitoon käytettävät merkkiaineet ovat kliiniseltä suorituskyvyltään puutteellisia. Verestä mitattavat merkkiaineet ovat usein glykoproteiineja, joiden glykaanirakenteet muuttuvat syövän kehittyessä. Glykaanirakenteiden muutokset eivät ole satunnaisia, vaan luonnonvalinta pienoisympäristössä suosii tiettyjä glykaaneja, jotka tarjoavat syöpäsoluille valintaedun. Syöpäspesifisten glykaanirakenteiden havaitseminen voisi johtaa kliiniseltä suorituskyvyltään entistä parempien syöpämerkkiaineiden kehitykseen. Tässä työssä oli tarkoituksena kehittää yleisimmän suolistosyöpämerkkiaineen, karsinoembryonaalisen antigeenin, glykaanirakenteita tunnistava määritys. Määritys toimi perinteisen kaksipuolisen immunomäärityksen periaatteella, sillä erolla, että leimatun vasta-aineen sijasta käytettiin glykaanirakenteisiin sitoutuvin proteiinein, lektiinein, päällystettyjä Eu3+-nanopartikkeleja karsinoembryonaalisen antigeenin glykaanirakenteiden havaitsemiseen. Kehitettyä lektiinimääritystä verrattiin karsinoembryonaalisen antigeenin kokonaispitoisuutta mittaaviin määrityksiin verinäytteissä (n=44). Määritysten välillä oli suuria eroja, varsinkin alhaisen karsinoembryonaalisen antigeenin kokonaispitoisuuden omaavissa näytteissä, jotka ovat ongelmallisia arvioida kokonaispitoisuuteen perustuvissa määrityksissä. Vaikka glykaanirakenteita tunnistava määritys voisi teoriassa olla kliiniseltä suorituskyvyltään kokonaispitoisuutta mittaavaa määritystä parempi, pitäisi sen tulokset yhdistää muihin merkkiaineisiin, mikäli sen avulla halutaan todeta varhaiset syöpätapaukset, sillä kaikki suolistosyöpäkasvaimet eivät eritä karsinoemryonaalista antigeeniä. Työssä onnistuttiin kehittämään kaksi karsinoembryonaalisen antigeenin glykaanirakenteita tunnistavaa määritystä, joiden mahdollisuudet kliinisiä sovelluksia ajatellen ovat kiinnostavat, mutta jatkotutkimuksia suuremmalla ja paremmin karakterisoidulla potilaspaneelilla tarvitaan määritysten kliinisen suorituskyvyn arvioimiseksi.Siirretty Doriast