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CancerPreventionResearchThe Forefront of Prevention Science

January 2015Volume 8Number 1

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Research Article

A Multiplexable, Microfluidic Platform for theRapid Quantitation of a Biomarker Panel for EarlyOvarian Cancer Detection at the Point-of-CareBasil H. Shadfan1, Archana R. Simmons1,2, Glennon W. Simmons1,2, Andy Ho1,2,Jorge Wong1,2, Karen H. Lu3, Robert C. Bast Jr4, and John T. McDevitt1,2

Abstract

Point-of-care (POC) diagnostic platforms have the potentialto enable low-cost, large-scale screening. As no single biomark-er is shed by all ovarian cancers, multiplexed biomarker panelspromise improved sensitivity and specificity to address theunmet need for early detection of ovarian cancer. We haveconfigured the programmable bio-nano-chip (p-BNC)—a mul-tiplexable, microfluidic, modular platform—to quantify a nov-el multi-marker panel comprising CA125, HE4, MMP-7, andCA72-4. The p-BNC is a bead-based immunoanalyzer systemwith a credit-card–sized footprint that integrates automatedsample metering, bubble and debris removal, reagent storageand waste disposal, permitting POC analysis. Multiplexed p-BNC immunoassays demonstrated high specificity, low cross-reactivity, low limits of detection suitable for early detection,and a short analysis time of 43 minutes. Day-to-day variability,

a critical factor for longitudinally monitoring biomarkers, ran-ged between 5.4% and 10.5%, well below the biologic variationfor all four markers. Biomarker concentrations for 31 late-stagesera correlated well (R2 ¼ 0.71 to 0.93 for various biomarkers)with values obtained on the Luminex platform. In a 31 patientcohort encompassing early- and late-stage ovarian cancers alongwith benign and healthy controls, the multiplexed p-BNC panelwas able to distinguish cases from controls with 68.7% sensitivityat 80% specificity. Utility for longitudinal biomarker monitoringwas demonstrated with prediagnostic plasma from 2 cases and 4controls. Taken together, the p-BNC shows strong promise as adiagnostic tool for large-scale screening that takes advantage offaster results and lower costs while leveraging possible improve-ment in sensitivity and specificity from biomarker panels. CancerPrev Res; 8(1); 37–48. �2014 AACR.

IntroductionIn 2013, an estimated 22,000 women in the United States were

diagnosed with ovarian cancer and 14,000 women died from thedisease (1). Although ovarian cancer is not one of the mostcommon forms of cancer, it has one of the highest case-to-fatalityrates, possibly due to the lack of distinctive symptoms in the earlystages of disease (2). Superior surgical management combinedwith carboplatin and paclitaxel chemotherapy have improvedtreatment, but there is still less than a 30% cure rate overall (3).Significantly, when the disease is detected in early stage (stage I),survival rates up to 90% can be achieved (4). However, only 20%to 25% of cases are diagnosed at an early stage (5). In addition,

there is no screening test currently recommended for the generalpopulation at average risk. Given the low prevalence of ovariancancer in postmenopausal women (1 in 2,500), any screening testfor ovarian cancermustmaintain a high specificity (99.6%)with asensitivity >75% for preclinical disease to achieve a positivepredictive value (PPV) of 10% (i.e., 10 operations per case ofovarian cancer detected) (4).

Transvaginal sonography (TVS) has been evaluated for earlydetection of ovarian cancer, but its poor specificity prompts 30operations for each case of ovarian cancer diagnosed (6, 7). Theblood biomarker Cancer Antigen 125 (CA125) has been used inclinics for more than three decades to monitor patient response totreatment and to detect ovarian cancer recurrence (8). Despite theclear correlation of elevated CA125 levels to the growth of manyovarian cancers, clinical utility for early detection remains to beestablished (5, 9). A two-stage screening strategy that utilizes risingCA125 to prompt TVS in a small fraction of women is beingevaluated by the United Kingdom Collaborative Trial of OvarianCancer Screening (UKCTOCS) and a Normal Risk Ovarian CancerScreening Trial in the United States (10–12). Both trials indicatethat no more than three operations will be required to detect eachcaseofovarian cancer (11, 12).Although the specificity is adequate,it remains to be seenwhether adequate sensitivity can be achieved.Early-stage disease has been detected in both trials, but the two-stage screeningmethod's impact on survival andmortality will notbedetermineduntil the conclusionofUKCTOCS trial in2015.Onepotential limitation of the initial stage of this strategy is that CA125isonlyelevated in sera from50%to60%ofwomenwithearly-stageovarian cancer at the time of conventional diagnosis and only

1Department of Chemistry, Rice University, Houston, Texas. 2Depart-ment of Bioengineering, Rice University, Houston,Texas. 3Departmentof Gynecologic Oncology,The University of Texas MDAnderson Can-cer Center, Houston, Texas. 4Department of Experimental Therapeu-tics, The University of Texas MD Anderson Cancer Center, Houston,Texas.

Note: Supplementary data for this article are available at Cancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

A.R. Simmons and G.W. Simmons contributed equally to this article.

Corresponding Author: John T. McDevitt, Chair, Department Biomaterials &Biomimetics, New York University College of Dentistry, 433 First Avenue, Room820 New York, NY 10010-4086, USA. Phone: 212-998-9204; Fax: 212-995-4244;E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-14-0248

�2014 American Association for Cancer Research.

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around 80%of all ovarian cancers express CA125 at the tissue level(5). To overcome the limited sensitivity of CA125, several multi-marker panels have been proposed that improve sensitivity com-pared with CA125 alone (13, 14). Improved sensitivity, however,needs to bemaintainedwithout compromising the high specificityof CA125, critical to achieve the minimum PPV required for earlydetection. In addition to detecting cancers that fail to expressadequate quantities of CA125, additional biomarkersmight detectdisease earlier thanCA125, conferring greater lead time (15). Froman assessment of 96 biomarkers utilizing xMAPbead-based immu-noassay technology, several multi-marker panels were identifiedfor early detectionofovarian cancer (16). Themostpromising eightmarkers of the study were tested further using ELISA and afterevaluating sensitivity, specificity, and biologic variability, a com-bination of CA125, HE4, MMP-7, and CA72-4 was chosen as themost promising panel with the highest sensitivity and specificity(unpublished data). Each of these biomarkers has been linked toabnormal function of ovarian cancer cells. CA125 has been impli-cated incell adhesion(17,18),HE4 inovarian cancer cellmigrationand adhesion (19, 20), MMP-7 in degrading extracellular matrixproteins in invasionand spreadof cancer cells (21,22), andCA72-4in inducing transformation of cells and tumorigenicity (23).

Multiplexing strategies for protein biomarkers have been cre-ated and commercialized with success. The most widely usedmethod is currently the Luminex flow cytometric bead-basedsystem (24). Although currently available multiplexing strategiesare capable of analyzing multiple biomarkers concurrently andhave been used for biomarker discovery (16), they require sig-nificant laboratory infrastructure and in the case of ovarian cancer-specific multi-marker assays require an overnight incubation.Microfluidic multiplexing systems have been proposed to over-come these challenges by utilizing the inherent advantages oflower sample and reagent volume (25).Microfluidic strategies arealso amenable to a point-of-care (POC) format due to theireconomic use of sample and reagents, rapid assay times, andintegration of the systems required for immediate analysis (26).Electrochemical immunoassay strategies in the microfluidicregime have been applied to multiplexing ovarian cancer bio-markers andhave demonstrated good analytical performance, butlong incubation times (10 hours) preclude the use of thismethodat the POC (27). Another proposed POC platform for ovariancancer is incapable of multiplexing and suffers from a limiteddynamic range despite 1-hour analysis times (28).

Multiplexing biomarkers in a rapid, POC diagnostic test thatcan quantitate the clinically relevant range of values has thepotential to revolutionize the use of biomarkers in clinical prac-tice. In the last decade, our laboratory has continued to evolve theprogrammable bio-nano-chip (p-BNC) for POC diagnostics(29, 30). The programmability of the agarose bead sensors com-bined with the customizable array allows for a wide-range ofmultiplexed panels for disease detection or overall health classi-fication (31, 32). The agarose bead-based immunoanalyzers aremade in-house and incorporated in credit-card sized disposablecards that integrate multiple on-card processes required for anal-ysis, including waste-containment, and are designed to interactwith an in-house, in-development portable reader containing allfluid actuation, optics and analysis software to rapidly quantitateprotein biomarker levels, permitting analysis at the POC (33).

A POCdiagnostic platform such as the p-BNC that can quantifymultiplex biomarker panels may aid the first-line of screening forovarian cancer and permit immediate TVS without the need to

schedule a second appointment. Such a strategymay enable rapidand often more cost-effective means of analysis for large-scalescreening methodologies, bypassing the need to transfer samplesto a remote clinical laboratory (34). By decreasing the lag-timebetween sample analysis, results, and possible referral to TVS,fewer patient visits will be required, reducing anxiety, travel time,and associated costs. These projected reduced costs have thepotential to improve screening methods such as Risk of OvarianCancer Algorithm (ROCA) by making 3-month screenings morefeasible (35, 36).

Similar to ROCA, levels of multiple biomarkers can be mon-itored over time to account for permanently elevated readingsfromunrelated conditions rather than using a strict cutoff value toindicate risk of disease. A custom baseline for each biomarker iscreated for individual patients rather than using an average,effectively creating a personalized cutoff range that when brea-ched indicates elevated risk (37). The potential improvement ofusing time-point data for all four biomarkers in a single algorithmis still being investigated with UKCTOCS patient samples at MDAnderson Cancer Center, but proteins other than CA125 may beelevated earlier in certain types of ovarian cancers, demonstratinganother advantage of a multiplexable screening method.Although longitudinally monitoring values has shown promisein helping with early detection, additional attentionmust be paidto the precision of the diagnostic system. Here, the assay/systemvariations must be significantly lower than the biomarker varia-tions within and between individuals to permit longitudinalmonitoring.

In this study, we program the p-BNC for high sensitivity, lowcross-reactivity multiplex analysis of a biomarker panel compris-ing CA125, HE4, MMP-7, and CA72-4. We explore the analyticalperformance of the system and demonstrate clinical applicationof the multiplex p-BNC for ovarian cancer sera of various stagesalong with healthy controls. We further explore the utility of themultiplexed p-BNC to assess longitudinal preclinical plasma ofwomen that develop ovarian cancer.

Materials and MethodsImmunoreagents preparation

All reagents (capture and detection antibodies, antigen) used inp-BNC immunoassays were prepared with SuperBlock (PBS)BlockingBuffer (ThermoFisher Scientific Inc.) to limit nonspecificbinding and enhance reagent stability. To develop heterologousdouble determinant immunoassays in the p-BNC format, murinemonoclonal anti-human CA125 (clone M11), anti-human HE4(clone 2H5), and anti-human CA72-4 (clone CC49) antibodieswere used to capture CA125, HE4, and CA72-4, respectively. Fordetection of captured ligands, anti-CA125 (clone OC125), anti-HE4 (clone 3D8), and anti-CA72-4 (clone B72.3) murine mono-clonal antibodies were used. Each of these reagents was gener-ously supplied by Fujirebio Diagnostics, Inc. Tomeasure MMP-7,murine monoclonal anti-MMP-7 (clone 111433) was used tocapture the ligand and goat anti-MMP-7 polyclonal antibody wasused for detection. Anti-MMP-7 antibodies were purchased fromR&D Systems, Inc. Capture antibodies were anchored on glyoxy-lated agarosemicrospheres manufactured in our laboratory usingpreviously reported reductive amination protocols at a concen-tration of 320 ng per agarose bead sensor (32). Primary aminogroups of detecting antibodies were conjugated to TFP estermoieties of appropriate fluorophores—AlexaFluor 488 (for HE4,

Shadfan et al.

Cancer Prev Res; 8(1) January 2015 Cancer Prevention Research38




MMP-7, and CA72-4 antibodies) and Oregon Green 488 (forCA125 antibody) to form stable dye-antibody conjugates(Thermo Fisher Scientific Inc.). Immunoassay standards for cal-ibration curves were prepared by generating appropriate dilutionsusing purifiedprotein antigen stocks forCA125,HE4, andCA72-4(Fujirebio Diagnostics Inc.) and EIA antigen standard for MMP-7(R&D Systems Inc.).

Programmable bio-nano-chip card fabricationThe p-BNCwas constructedwith alternate layers of 3M9500PC

double-sided adhesive (3M Company) and 3M AF4300 polyeth-ylene terephthalate (3M Company), which were patterned xuro-graphically with a SummaCut D75 (Summa Inc.) and a GraphtecFC2250 plotter cutter (Graphtec America, Inc.), respectively. Theresulting seven-layer card featured a network of microfluidicchannels for sample delivery and metering, detecting antibodyreconstitution and delivery, andwashing. Air vents were built intothe card using hydrophobic SurePVDF membranes (EMD Milli-pore) to mitigate bubbles and an 8-mm Whatman NucleporeTrack-Etch membrane (GE Healthcare) was used for in-line fil-tration. Glass fiber conjugate pads (EMD Millipore) cut into 2 �15mmrectangleswereused to store detecting antibody in the cardand mixers were patterned into the polyethylene terephthalatelayer's channels with theGraphtec plotter cutter to ensure homog-enous mixing and consistent delivery of fluids across bead array.The 4 � 5 array, designed with hexagon-shaped wells to localizeindividual agarose bead sensors, was cast in an UV-curable pho-topolymer (Norland Products Inc.) from a custom machinedaluminum mold. The array was embedded in the card and wassealed with a cyclo olefin polymer thermoplastic plastic cover(Zeon Corp.) to permit optical access following functionalizationwith agarose beads on which the sandwich immunoassays werecompleted. Design rationale and engineering optimization thatled to the final design will be reported elsewhere.

Assay execution and image analysisAll assays were performed using the in-house xurography-

patterned microfluidic card as described in the previous section,using NE-1000 syringe pumps (New Era Pump Systems Inc.) tovariably control fluid flow. A volume of 12 mL of detectingantibody cocktail containing 0.15 mg of CA125, 0.15 mg of HE4,0.6 mg of CA72-4, and 0.015 mg of MMP-7 detecting antibodiesprepared in SuperBlock (PBS) Blocking Buffer was depositedonto the glass fiber pad. Agarose beads functionalized withcapture antibody specific to the analyte of interest (CA125,HE4, MMP-7, or CA72-4) were localized to predeterminedwells to permit spatial identification and consequently multi-plexing. Four redundant sensor beads per analyte permittedelimination of agarose beads with visual signal obstruction andenabled evaluation of within-assay variance. In addition to thebead sensors for analytes, two negative control beads coupledwith immunoglobulin G (IgG) for irrelevant analytes (IL1-b/cTnI antibodies) and two positive control beads (goat anti-mouse IgG antibodies) were localized to the wells. Followinglocalization of functionalized beads and corresponding con-trols, 100 mL of sample was loaded into each card through thesample entry port, which was then sealed using a double-sidedadhesive cover. For clinical samples, 50 mL of sample wasdiluted with 50 mL of SuperBlock (PBS) Buffer and the subse-quent 100 mL was loaded into the card for analysis. Twosyringes of SuperBlock (PBS) Buffer were connected to the two

main entry ports of the card to supply pressure driven flowinside the card. By actuating the first pump, the 100 mL samplewas filtered in-card and passed over and through the beads for aperiod of 30 minutes. After a 1-minute rinse at 100 mL/min towash unbound antigen, the detecting antibody was elutedfrom the pad and introduced to the bead array at the rate of10 mL/min with the second syringe. After 7 minutes, a final rinseof 100 mL/min for 5 minutes was utilized to wash unbounddetecting antibody. The total assay time was 43 minutes with atotal assay volume of 770 mL.

Images were acquired using �10 magnification on a modifiedOlympus BXFM epifluorescent microscope and analyzed usingImageJ (NIH) with previously described custom image analysismacros to determine the mean fluorescence intensity along theperimeter of the bead (38). Optical obstruction of the agarosesensor beads via bubbles or sample/reagent debris resulted in therejection of the bead sensor for quantitative analysis.

Calibration curves were generated and fitted to a standard four-parameter logistic regression with SigmaPlot 12.0 (Systat Soft-ware, Inc.) software. Unknown concentrations for samples wereinterpreted from the standard curve using the same software.Microsoft Excel (Microsoft Corp.) was used to analyze precisionstudy data andMedCalc (MedCalc Software) was used to generatereceiver operating characteristics (ROC) curves and box plots forclinical sample concentrations.

Precision studyHuman serum-based Liquichek Tumor Marker Controls (Bio-

Rad Laboratories) of three different concentrations (low, medi-um, and high range of each analyte) were assessed on the p-BNCin triplicate over 3 days to assess within-day and between-dayvariation of the multiplexed assay on the p-BNC. CA125 isincluded in the manufacturer's preparation of the controls andcorresponding antigen levels of HE4, CA72-4, and MMP-7 werespiked in each of the three levels (concentrations reported inSupplementary Table S1). The intra-assay variationwas defined asthe variation between redundant bead sensors in a single assayrun, whereas the inter-assay precision was defined as the coeffi-cient of variation between different runs over separate days. Thecoefficient of variation was averaged across the three concentra-tion levels to report the final inter- and intra-assay precision.

Clinical samplesPlasma and serum samples were collected and stored at�80�C

at the MD Anderson Cancer Center (Houston, TX) gynecologiconcology tumor bank following standard IRB-approved proto-cols. Three independent banked sample sets were used in thisstudy, totaling 95 serum samples from 68 patients. For methodvalidation, sera from 31 advanced stage (III–IV) patients wereassayed. To evaluate the clinical performance of the p-BNCmulti-plexed system, sera fromhealthy individuals (n¼7), patientswithbenign gynecologic conditions (n ¼ 8), patients with early-stageovarian cancer (n¼ 7), and patients with late-stage ovarian cancer(n ¼ 9) were assayed. To test the potential of the p-BNC to assaysequential samples from the same women, plasma samples wereassayed that had been collected at multiple (2–12) time pointsfrom women who had remained healthy (n ¼ 4) and who haddeveloped ovarian cancer (n ¼ 2) during the course of the study.Samples were acquired from the MD Anderson Normal RiskOvarian Cancer Study (NROS) serum bank (12). All sampleswere thawed at 4�Cbefore use and diluted two-fold in SuperBlock

p-BNC Multiplexed Detection of Ovarian Cancer Biomarkers

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(PBS) Buffer before analysis on the p-BNC. For method valida-tion, sera were measured on custom multiplex assays for CA125,HE4, MMP-7, and CA72-4 (with CA125 and MMP-7 assays fromEMD Millipore multiplexed with CA72-4 and HE4 assays devel-oped in-house) at MD Anderson Cancer Center on the LuminexMAGPIX magnetic bead-based immunoassay system.

ResultsThe p-BNC assay card (Fig. 1) is a lab-on-a-chip platform that

features a bead-based sensor core and a fully integrated micro-fluidic network that facilitates on-card sample preparation andmetering, reagent storage, mixing, bubble and debris removal,and secure waste containment. By design, the p-BNC assay cardminimizes benchtop sample and reagent preparation steps andassociated laboratory tools and infrastructure, which is critical forPOC analysis. Agarose beads represent the sensor core on whichthe sandwich immunoassays occur for quantification of biomark-er analytes (Fig. 1B). The supporting microfluidic environment,coupledwith the 3D structure of the agarose beads permit optimalanalyte capture and detection to achieve low limits of detectionand short analysis times. Further, due to the predefined spatialarrangement of the beadson the chip (Fig. 1C), the p-BNCpermitsmultiplexing of several biomarkers together. Following sampleinput, the sample is automatically metered on the card andprocessed to remove bubbles and debris. The buffer reagentspush the sample to the beads, permitting analyte molecules tobe captured on the immunosensor (antigen delivery) followed byremoval of unbound analytes (wash step). The detecting antibodystored on the glass fiber pad is then eluted by the buffer anddelivered to the beads to complete the immunoassay sandwichand is followed by a wash step before signal capture. All elementsof the card, including the footprint, were designed in parallel witha portable optical analyzer to ensure rapid translation of thedevice to a fully portable POC assay system.

In this work, we configure a four biomarker panel comprisingCA125, HE4, MMP-7, and CA 72-4 to the p-BNC. This biomarkerpanel has been discovered as an optimal panel in distinguishingearly-stage ovarian cancer patients from corresponding controlsresulting fromdiscovery efforts atMDAndersonCancerCenter. Asbiomarkers are added or removed the p-BNC system can rapidlybe reconfigured by replacing, adding, or removing bead-types inthe array, and therefore reduce the time frombiomarker discoveryto endpoint analysis.

Assay optimizationTo develop POC-suitable assays for the multiplex panel, the

assay rangeswere defined on the basis of clinical requirements. Anassay suitable for early detection biomarkers in a POC settingshould be capable of measuring the relevant range of biomarkersin a short period of time. The dynamic range to encompass theearly detection and healthy range for individuals for all fourbiomarkers were defined on the basis of previous work (16). Tofunction in a POC setting, a total assay time of no longer than 1hour was set as the upper limit to include sample incubation,detecting antibody delivery, and wash steps to remove unboundantigen and detecting antibody. To measure the four biomarkerson the p-BNC within 1 hour over a dynamic range with smallvolumes of sample, extensive optimizationwas required. The fourbiomarker assays were developed individually, beginning withthe identification of an optimal matched pair. On the basis ofprevious work, an optimal concentration of 320 ng/bead ofcapturing antibody was coupled to the beads to permit high-sensitivity assays (30). A 30-minute sample (antigen) incubationtime was chosen for delivery to permit adequate analyte capturewhile maintaining the short assay times required for a POC assay.A 7-minute incubation with detecting antibody was chosen topermit sufficient time to complete the sandwich immunoassaywithout increasing background. Flow rates were also optimized to

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Figure 1.Illustration of (A) disposable p-BNC card with (i) syringe pump flow adapters, (ii) sample entry port and (iii) bead array holder. The (iv) sample loop contains an (v)overflow chamber, ensuring a 100 mL dose of sample for each card. The right pump adapter flows buffer into the sample channel, displacing the sample intothe bead-holding chip and through the beads into the (vi) waste. The second pump elutes stored detecting antibody off the (vii) glass fiber pad and into the chip. Theflow rate of the second pump is increased to remove the unbound reagents and to perform the final rinse for imaging. All fluid pumped into the card passesthrough (viii) bubble traps to remove bubbles and an 8 mm filter to prevent debris from reaching the bead sensors. A molecular schematic of the agarose bead isshown in (B), demonstrating a completed sandwich immunoassay. (C) shows a zoomed-in illustration of the bead array holder with different colors indicatingdifferent bead sensor types permitting multiplexing based on spatial identification.

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permit proper antigen interactionwith the capture antibodies (3.3mL/min), adequate delivery time, and consistent dosing of detect-ing antibody (10 mL/min), and to assure sufficient washingwithout dislodging or destroying bead sensors (100 mL/min).Detection antibody optimization involved evaluation of differentdilutions fromundiluted to a 1:40 dilution.Higher dye loading ofthe detecting antibody than recommended by manufacturer'sprotocols (achieved by overnight incubation rather than 1 hourincubation) resulted in significantly increased signal without acorresponding increase in background noise for detection anti-bodies. This strategy increased the number of fluorophore moi-eties per antibody and was found to be suitable for CA125 andCA72-4 assays. Higher fluorescent intensity at a given signal wasachieved with AlexaFluor 488 for HE4, CA72-4, and MMP-7antibodies, whereas Oregon Green 488 outperformed AlexaFluor488 for CA125. In addition, a �10 magnification significantlyimproved signal-to-noise ratio (SNR) over the previously used�4magnification for the p-BNC, due to the advantage derived fromthe higher numerical aperture. In addition, a background sub-traction method was used in ImageJ to reduce background noise

and further improve the SNR. These optimization steps werenecessary to achieve the performance required for the individualimmunoassays. A combination of these optimization stepsresulted in performance suitable for POC time constraints andearly detection assay range.

Analytical performance and validationFollowing optimization as described above, the dose–

response curves for the resulting multiplexed immunoassaysfor each of the four individual markers, fitted to a four-param-eter logistic equation are shown in Fig. 2. Standard curves weregenerated by serial dilution of a standard concentration in amultiplex cocktail of the four antigen standards, encompassingranges required to cover the biomarker range of interest(healthy women and patients with early stage ovarian cancers):for CA125, 0–400 U/mL; HE4, 0–200 pmol/L; CA72-4, 0–50U/mL; and MMP-7, 0–10 ng/mL. The multiplexed limits ofdetection, evaluated as the concentration on the four-parameterlogistical equation corresponding to three standard deviationsabove zero, for the four markers are shown in Table 1 and

Figure 2.Calibration curves showing variation of mean fluorescent intensity (MFI) across concentration ranges tested for (A) CA125, (B) HE4, (C) MMP-7, and (D)CA72-4 obtained on the p-BNC in amultiplexed format andfitted to four-parameter logistic regression curves. Error bars designate the inter-assay variation betweenanalyte-specific beads. The calibration curves indicate suitability for measurement for low concentrations of biomarkers found in healthy and early disease stateswith low intra-assay variation across the entire range of measured concentrations. Plots of the lower range of each biomarker dose curve with alinear fit are included in Supplementary Fig. S1.






displayed in the lower range of each dose–response curve inSupplementary Fig. S1. All four assays are capable of reliablemeasurements below the cutoff values established for healthyindividuals and reported in literature: CA125, 35 U/mL(12); HE4, 40–85 pmol/L (age-dependent; ref. 39); CA72-4,8.5 U/mL (40); and MMP-7, 7.4 ng/mL (41). Reliable measure-ments of biomarker concentrations below cutoff levels areessential for establishing baselines for longitudinal biomarkermonitoring. The intra-assay precision that measures variationbetween bead-sensors for a given analyte and the inter-assayprecision that measures the variation between independentassays for a given analyte were evaluated for three differentconcentrations encompassing the dynamic range of the assayand were averaged (Table 1). Intra-assay precision ranged from5.0% to 9.4%, whereas the inter-assay (conducted on 3 con-secutive days) precision ranged from 5.4% to 10.5% anddemonstrate low assay-specific variability. Longitudinal algo-rithms for biomarkers rely on within-person and between-person biologic variation and also the rise in biomarkers levelsfrom the baseline (36). The within- and between-assay preci-sion were both well below the biologic variation of the indi-vidual markers, determined previously through ELISA (manu-script in preparation).

Cross-reactivityArguably the most significant challenge with multiplex immu-

noassays is the presence of cross-reactivity between the detectingantibodies and capturing antibodies for the different analytes. Inan ideal multiplexed assay, only the capturing antibody anddetecting antibody specific to the analyte of interest will form animmunoassay sandwich, whereas interactions with nonanalyte-specific antibodies and antigen will be minimal. However, inreality, several nonideal interactions are possible between thereagent components of the various multiplex assays (42) that inturn can lead to decrease or increase in specific signal, which mayaffect the analytical performance of the multiplexed immunoas-say in comparison with the singleplex assay. In addition, cross-reactivity can also occur over the entire dynamic range, precludingmultiplexing of the candidates of interest. To minimize cross-reactivity, all multiplex reagents were chosen with the other threeanalytes serving as negative controls. If any signalwas observed onthe bead-sensors for the nonspecific analyte, other matched pairantibody configurations were explored.

In addition, while optimizing variables such as flow rates,volumes, and detecting antibody dilutions, all four bead-sensorswere used to select assay conditions that minimized cross-reac-tivity. To explore the cross-reactivity across the entire range ofanalyte concentrations, both the singleplex assays (with theanalyte, capturing antibody, and detecting antibody of interest)andmultiplex assays (with all four analytes and their correspond-ing capture and detecting antibodies) were completed. The cal-ibration curves for the singleplex and multiplex assays obtained

are shown in Fig. 3. The curves obtained from both methodsoverlap significantly, demonstrating that the signalsmeasured at agiven concentration are similar for both singleplex and multipleassays. Less than 10% variation was noted between the singleplexand multiplex dose responses demonstrating negligible cross-reactivity. The four detecting antibodies were combined on asingle reagent pad during all multiplexed assays, demonstratinginitial compatibility for long-term, in-card storage. Notably, theLODs observed post multiplexing were comparable with theindividual assay LODs, demonstrating that the assay performancewas retained upon multiplexing. Thus, reliable measurementsmay be obtained upon multiplexing without loss of assay per-formance and with comparable signal intensities and minimalcross-reactivity.

Clinical validationTo compare the clinical measurements obtained on the multi-

plexed p-BNC to laboratory standardmultiplexmeasurements onthe Luminex MAGPIX instrument, we measured sera obtainedfrom 31 late-stage ovarian cancer patients using both methodol-ogies. The method comparison plot (Fig. 4 and SupplementaryFig. S2) shows the concentrations obtained from both methodsfor a given sample, plotted against each other. The correlationcoefficient obtained for all four biomarkers ranged from 0.71 to0.93, demonstrating good correlation between the two methods.

Followingmethod validation, we chose to explore the ability ofthe multiplexed biomarker panel assessed on the p-BNC todistinguish ovarian cancer patients from patients with nonma-lignant gynecologic conditions and fromhealthy women. For thispurpose, we assayed 31 sera from 7 healthy women, 8 patientswith benign gynecologic conditions, 7 patients with early-stageovarian cancer, and 9 patients with late-stage ovarian cancer. Theresults obtained are shown as dot plots in Fig. 5, displaying theconcentrations of individual biomarkers in cases and controlsalong with suggested cutoff levels for the given cohort of samples.The specificity and sensitivity derived from utilizing these cutoffsare indicated beside the dot plots. Themultiplexed concentrationsobtained on the p-BNC are capable of distinguishing betweencases and controls. Also, the cutoff concentrations for theseindividual biomarkers obtained on the p-BNC are comparablewith previously reported values for these markers (12, 39–41). Inaddition, the specificities and sensitivities reported for the indi-vidual biomarkers for ovarian cancer are concordant with valuesreported in the literature. The values of all four biomarkers werecombined using a logistic regression model using the MedCalcstatistical software and the ROC curves for the multi-markercombination and the individual biomarkers are shown in Fig.6. The multi-marker combination shows improved performanceover individual biomarkers alone, performingwith a sensitivity of68.7% at 80.0% specificity, demonstrating the improved sensi-tivity from using multi-marker panels.

To explore the ability of the p-BNC to perform serial measure-ments of biomarkers over time, we chose a cohort of 6 individualsfrom whom blood plasma was collected annually over multipleyears. Of these 6 women, 2 developed ovarian cancer during thestudy period, whereas the other 4 did not develop cancer andwerehence deemed healthy. The biomarker profiles of these indivi-duals as obtained on the p-BNC are shown in Fig. 7. For each ofthese individual biomarkers, the healthy individuals have theirown baseline that remained flat during the course of the study. Inone of the two cases, the biomarker concentrations rose sharply in

Table 1. Multiplexed limits of detection (LOD) and intra- and inter-assayprecision values for CA125, HE4, MMP-7, and CA72-4

BiomarkerMultiplexLOD

Inter-assayprecision

Intra-assayprecision

CA125 1.8 U/mL 10.5% 9.4%HE4 2.3 pmol/L 5.4% 5.0%MMP-7 0.2 ng/mL 8.5% 8.7%CA72-4 1.7 U/mL 8.3% 9.1%

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A B

DC

E FCA125 HE4 MMP-7 CA72-4CA125 HE4 MMP-7 CA72-4

POSNEG

POSNEG

Figure 3.Calibration curves obtained for (A) CA125, (B) HE4, (C) MMP-7, and (D) CA72-4 in a singleplex (black circles) and multiplex (white circles) format. Akin toELISA, the singleplex experiments only used the antigen of interest and the corresponding antibody for the sandwich immunoassay, whereas the multiplexexperiments assayed a cocktail of all four antigens with a cocktail of all four detection antibodies. The multiplex assays for each of the four analytes aresuperimposable with the individual singleplex assays demonstrating negligible cross-reactivity from the introduction of other reagents due to multiplexing.Representative images (contrast enhanced) of a singleplex assay (E) and a multiplex assay (F), where the singleplex demonstrates specific signal for the analyte ofinterest (CA125) and the multiplex demonstrates specific signals for a cocktail of analytes.






comparison with the horizontal plots for the controls. In case 2(isolated in Supplementary Fig. S3), CA125 levels did not increasesubstantially, whereas the other three biomarkers rose beforediagnosis, demonstrating the importance of additional biomarkerinformation.

Although it is premature to determine clinical utility with thesmall sample cohort assessed, the studynonetheless demonstratesthe ability of the p-BNC to reliably measure biomarkers todistinguish disease states based on a multiplex panel and alsoshows strong promise for serial measurements of longitudinalbiomarkers. The four-biomarker panel is being validated withLuminex in 88 cases and 538 controls from the UKCTOCS trialand will be reported elsewhere.

DiscussionPOC diagnostic platforms have the potential to transform

clinical diagnostics by significantly reducing the turn-around timebetween sample analysis and biomarker results that can beinterpreted by the clinician. Microfluidic-based POC systems areinherently associated with low sample volumes and analysistimes and in the case of a system such as the p-BNC, also permitmultiplexing. Unlike traditional ELISAs,where only onebiomark-

er may be assessed, multiplexing offers additional advantages ofassessing multiple biomarkers simultaneously, thus reducingrequired sample volumes and analysis times and allowing incor-poration of multi-marker signatures. Particularly, in the case ofovarian cancer detection, this additional biomarker informationhas the potential to improve sensitivity while maintaining spec-ificity. Previously, we adapted the p-BNC for measurements ofCA125 in sera with analytic performance comparable with ELI-SAs, but with a shortened analysis time of 43minutes (30). In thiswork, we reduced the sample volume 10-fold and incorporatedadditional biomarkers from a novel ovarian cancer multi-markerpanel on the p-BNC to permit multiplexing. This multi-markerpanel, including CA125, HE4, MMP-7 and CA72-4, was chosenbased on sensitivity and specificity values, which will be pub-lished elsewhere after completion of validation. The p-BNCleverages the microfluidic regime to reduce assay time and costwhile enabling multiplexing and providing a path for a POCovarian cancer screening test.

Analytic performance of the multiplexed assay on the p-BNC demonstrated the ability of the platform to measure awide range of concentrations of CA125, HE4, MMP-7, andCA72-4 (Fig. 2), including values found in most healthy patientsamples. Cross-reactivity between the four assays was found to

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Figure 4.Correlation between the concentrations obtained on the p-BNC multiplex assays and Luminex MAGPIX multiplex assays for (A) CA125, (B) HE4, (C) MMP-7,and (D) CA72-4 for measurements in the 31 advanced-stage ovarian cancer sera. Plots demonstrate good correlation between the two methods. Plots omittinghigher value points and focusing on the lower range of values are included in Supplementary Fig. S2.

Shadfan et al.





be minimal, assuring comparable performance between the sin-gleplex and multiplex assays for the individual analytes (Fig. 3).Method validation using a cohort of late-stage ovarian cancerpatients showed good correlation for all four markers (R2 >0.71, Fig. 4)withflow-cytometricbead-based immunoassay system(Luminex), the current research laboratory standard for multiplex

immunoassays. In addition, apilot study cohort ofhealthy, benign,early-stage, and late-stage patients assessed on the p-BNC demon-strated the ability of the p-BNC to differentiate cases fromcontrols with acceptable specificity and sensitivity. All patientsamples required only 50 mL of sample per assay or 12.5 mL peranalyte.

The longitudinal monitoring of biomarkers could signifi-cantly improve diagnostic performance of biomarkers by cre-ating personalized baselines rather than using arbitrary cutoffsthat could result in higher false-positive rates (43). The cost-and time-reduction of POC tests could benefit a longitudinalmonitoring strategy such as ROCA by reducing two patientvisits (sample collection and TVS referral) to one and thereforemake 3-month screenings more feasible for patients with risinglevels of biomarkers. This strategy can also be applied to high-risk patients with familial history of ovarian or breast cancer orwith predisposing mutations such as BRCA1/2, demonstratedto need more frequent screenings in the United KingdomFamilial Ovarian Cancer Screening Study (44). The ability toprocess the patient sample and obtain clinically interpretableresults within a single visit will permit TVS on the same visit,avoiding anxiety and additional travel time. A POC test used forlongitudinal monitoring requires analytic precision (within-and between-assay) that is much lower than the biologicvariation of patient samples. Indeed, the p-BNC precision studydemonstrated lower analytic %CVs in comparison with thebiologic variation in healthy individuals for each of thesebiomarkers. In addition, assessment of longitudinal plasmafrom 2 ovarian cancer patients and 4 healthy controls demon-strated the use of the p-BNC for longitudinal monitoring.Clearly, all four individual biomarkers showed flat baselinesin healthy patients and elevated significantly following a periodof flat levels in one of the two cases. In the second case with lowCA125, additional biomarkers rose indicating the potential ofbiomarker complementarity permitted by use of a multi-mark-er panel.

Figure 5.Dot plots of (A) CA125, (B) HE4, (C)MMP-7, and (D) CA72-4 measuredfrom a set of clinical samplescontaining healthy and benign patients(controls, n ¼ 15) and early-stage andlate-stage ovarian cancer patients(cases, n ¼ 16). Cutoff levels for eachbiomarker were determined inMedCalc by achieving highestsensitivity (including as many casesabove cutoff as possible) withoutsacrificing significant specificity(including as many controls belowcutoff as possible) and are shown bythe horizontal line in each plot. Thesedot plots demonstrate the clinicalperformance of the p-BNC todistinguish between variousdisease states.

Figure 6.ROC curves for CA125, HE4, MMP-7, CA72-4, and a combination of the fourusing a logistic regressionmodel. ROC curveswere determined from a clinicalsample cohort of 31 patients comprising healthy, benign, early-stage, andlate-stage ovarian cancer patients. Areas under the curve were 0.77 (CA125),0.70 (HE4), 0.55 (MMP-7), 0.71 (CA72-4), and 0.88 (combined) indicatingthat the multiplex panel outperformed the individual markers as assessed onthe p-BNC.






The panel used in this article has shown initial promise in earlydetection of ovarian cancer, but, importantly, the programma-bility of the p-BNC facilitates replacing, adding, or removingbiomarkers if better combinations emerge from clinical trials,which is crucial in the diagnosis of such a complex disease asovarian cancer, where the depth of knowledge of the field isconstantly expanding. Other classes of biomarkers such as auto-antibodies, nucleic acids or cells may also be assessed on the p-BNC (33), thus permitting the p-BNC to rapidly adapt to themostpromising discoveries in the field.

Parallel developments are under way to finalize a portableoptical instrument that will fully mobilize the p-BNC assay cardand enable analysis at the POC. The portable analyzer willreplace the bench-top epifluorescent microscope, syringepumps, and desktop computer used in this study. Further,progress has been made to translate the p-BNC assay card intoa more cost-effective injection-molded format, following bestpractices in design for manufacturing. The availability of injec-tion-molded disposables in this area has potential to acceleratethe translation of the assay system to real world application. Inaddition, the consistency resulting from mass manufacturing ofinjection-molded cards should serve to lower imprecisioncompared with the in-house made cards used in this study.The assay conditions derived in this work have served indeveloping the technical specifications for these advancementsof the system.

In this study, a POC-amenable microfluidic platform wasadapted for rapid analysis of a multiplex biomarker panel withlow detection limits and high precision with analysis timesshorter than comparable commercial methods. With a shortanalysis time and inclusion of novel markers for early ovariancancer detection, this platform shows strong promise as apotential POC screening method for ovarian cancer, wherepatients could receive results promptly enough to be referredto TVS in the same visit. Reduced costs and easier accessibilityto results could also assist in longitudinally monitoring bio-marker values over time, which has shown some promise inhelping detect early-stage ovarian cancer.

Disclosure of Potential Conflicts of InterestR.C. Bast Jr has ownership interest (including patents) in Fujirebio Diag-

nostics Inc and is a consultant/advisory board member for Vermillion. J.T.McDevitt has ownership interest (including patents) in and is a consultant/advisory boardmember for SensoDx LLC.No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: B.H. Shadfan, A.R. Simmons, G.W. Simmons, K.H. Lu,R.C. Bast Jr, J.T. McDevittDevelopment of methodology: B.H. Shadfan, A.R. Simmons, G.W. Simmons,J.T. McDevittAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B.H. Shadfan, A.R. Simmons, A. Ho, K.H. Lu

Figure 7.Longitudinal plasma from 6 women with multiple time-points of sample collection for each patient were measured on the p-BNC and values for (A) CA125,(B) HE4, (C) MMP-7, and (D) CA72-4 are shown over time. Two women (case 1 and 2) developed ovarian cancer and four other women (controls 1–4) did not. Thelongitudinal profiles show rise in biomarker concentrations for cases in comparison with flat profiles for controls. The time point in years of each measurement islisted in Supplementary Table S2.

Shadfan et al.





Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B.H. Shadfan, A.R. Simmons, A. Ho, R.C. Bast JrWriting, review, and/or revision of the manuscript: B.H. Shadfan, A.R. Sim-mons, G.W. Simmons, A. Ho, J. Wong, K.H. Lu, R.C. Bast Jr, J.T. McDevittAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B.H. Shadfan, K.H. Lu, R.C. Bast JrStudy supervision: A.R. Simmons, K.H. Lu, R.C. Bast Jr, J.T. McDevittOther (designing of the disposable assay cartridge used in the study): G.W.SimmonsOther (figures concept design): J. Wong

AcknowledgmentsThe authors thank Fujirebio Diagnostics, Inc., for generously providing

antibodies and antigen. The authors also thank Joseph Celestino for assistancein obtaining clinical samples from the MD Anderson tumor bank.

Grant SupportJ.T. McDevitt acknowledges support from the Cancer Prevention and

Research Institute of Texas (CPRIT; RP101382) and the National CancerInstitute (NCI) Ovarian Cancer SPORE (P50 CA 83639). K.H. Lu and R.C.Bast Jr acknowledge support from the MD Anderson CCSG NCI P30CA16672, the Mossy Foundation, Golfers Against Cancer, and Stuart andGaye Lynn Zarrow.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 1, 2014; revised September 24, 2014; accepted October 29,2014; published OnlineFirst November 11, 2014.

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2015;8:37-48. Published OnlineFirst November 11, 2014.Cancer Prev Res Basil H. Shadfan, Archana R. Simmons, Glennon W. Simmons, et al. Point-of-Carea Biomarker Panel for Early Ovarian Cancer Detection at the A Multiplexable, Microfluidic Platform for the Rapid Quantitation of

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