ovarian cancer is the 
 fifth leading cause of cancer - related mortality 
 of women in the u.s . , with over 15  000 deaths per year .
early diagnosis is associated with improved overall 
 survival ; however , the majority of patients 
 are currently diagnosed with advanced disease .
the 5 year survival 
 rate for late - stage ovarian cancer remains less than 30% . despite 
 the identification of serum ca 125 as a biomarker for ovarian cancer 
 in 1983 , there are currently no screening 
 biomarkers recommended for use for the general population .
the 
 utility of ca 125 as a screening test is limited by a low sensitivity 
 of 50% for early stage disease at 99% specificity . combining ca
125 with transvaginal ultrasound ( tvus ) increased 
 the specificity of detection in the ukctocs large - scale screening 
 trial . in a joint validation study of 
 28 potential markers for detecting ovarian cancer in blood ,
panels of markers demonstrated 
 only marginal improvements over ca 125 alone for the early detection 
 of disease .
a recent study showed that the addition of cea and vcam-1 
 to ca 125 and he4 increased the sensitivity of detection of stages 
 i and ii ovarian cancer to 86% at 98% specificity , but this remains to be confirmed in a blinded validation 
 study using prediagnostic sera .
protein overexpression or mutation can lead 
 to the spontaneous 
 development of autoantibodies ( aab ) in the sera of patients with cancer .
tumor antigen - specific aab have been identified 
 in the sera of patients with cancer , including patients with early 
 stage disease .
there are several potential 
 advantages of aab biomarkers , including signal amplification by the 
 immune response and persistence of antibodies after antigen is no 
 longer detected .
p53-specific aab , which are associated with p53 mutation 
 and resultant protein stabilization , have been detected in early stage 
 ovarian cancer .
we detected p53-specific 
 aab in 41.7% of patients with serous ovarian cancer at 91.7% specificity . unlike ca 125 and he4 , p53-aab were associated 
 with improved survival ( hr = 0.56 ) .
we hypothesized that the identification of novel aab biomarkers 
 beyond p53-aab would increase the sensitivity of detection of serous 
 ovarian cancer .
we used the custom protein microarray technology nucleic 
 acid protein programmable arrays ( nappa ) , which are generated by printing 
 full - length cdnas encoding the target proteins at each feature of 
 the array .
the proteins are then transcribed and translated by a cell - free 
 system and immobilized in situ using epitope tags fused to the proteins .
nappa arrays have been used to identify 
 antibody signatures in early stage breast cancer .
the objective of this study was to identify novel aab biomarkers 
 for the detection of serous ovarian cancer . to profile the ovarian 
 cancer immune response , we developed protein microarrays displaying 
 5177 full - length candidate antigens .
we used a sequential screening 
 strategy to select candidate aab biomarkers to limit the false discovery 
 rate inherent to large - scale proteomic screening .
first , we screened 
 34 cases of serous ovarian cancer and 30 matched healthy controls 
 ( cohort 1 ) on all 5177 candidate tumor antigens and selected 741 antigens 
 for further testing .
second , we screened 60 cases and 60 healthy controls 
 ( cohort 2 ) on the 741 antigens and identified 12 potential candidate 
 aab biomarkers .
third , we used an independent assay ( luminex bead 
 array ) to display these autoantigens and rescreened sera from women 
 in cohort 2 .
finally , we displayed a smaller set of 7 autoantigens 
 and screened sera from an independent set ( cohort 3 ) of non - serous 
 cancers ( n = 30 ) , false - negative ca 125 ( n = 20 ) , benign ovarian disease ( n = 15 ) , 
 and healthy controls ( n = 15 ) . the sensitivity and 
 specificity of each individual biomarker , as well as the biomarker 
 panel , are presented .
sera 
 used in these analyses were obtained 
 from brigham and women s hospital and the dana - farber cancer 
 institute with support from the nci early detection research network 
 and ovarian spore program .
sera derived from ovarian cancer patients 
 were obtained at the time of presentation prior to surgery , and patients 
 received routine postoperative therapy ( as described in ref ( 12 ) ) .
the non - serous cases were derived from 
 10 patients with endometrioid cancer , 10 patients with clear cell 
 carcinoma , and 10 patients with mucinous carcinoma .
the benign disease 
 samples were derived from 19 patients with serous cytadenomas and 
 11 patients with mucinous cystadenomas .
sera from age - matched general 
 population control women were obtained from brigham and women s 
 hospital using a standardized serum collection protocol and stored 
 at 80 c until use . cases and matched controls were processed 
 simultaneously .
women with a personal history of cancer ( other than 
 non - melanoma skin cancer ) were excluded as controls . written consent 
 was obtained from all subjects under institutional review board approval .
sequence - verified , 
 full - length cdna expression plasmids in flexible 
 donor vector systems were obtained from the arizona state university 
 biodesign institute and are publicly available ( www.dnasu.org ) .
these were converted to the t7-based mammalian expression vector 
 pant7_gst using lr recombinase ( invitrogen , carlsbad , ca ) .
the high - throughput 
 preparation of high - quality supercoiled dna for cell - free protein 
 expression was performed as described . for bead array elisas ,
larger quantities of dna were prepared using 
 standard nucleobond preparation methods ( macherey - nagel inc . ,
plasmid dna ( 1.5 g / ml ) , capture antibody 
 ( 50 g / ml anti - gst antibody , ge healthcare biosciences , piscataway , 
 nj ) or anti - flag antibody ( sigma - aldrich , st .
louis , mo ) , protein 
 cross - linker ( 2 mm , bs3 , pierce , rockford , il ) , and bsa ( 3 mg / ml , 
 sigma - aldrich ) were co - printed onto the array surface . all samples 
 were printed using a genetix qarray2 with 300 m solid tungsten 
 pins on amine - treated glass slides . the printed dna was transcribed 
 and translated in situ using reticulocyte lysate according to previously 
 published protocols .
protein expression 
 was detected using anti - gst mab ( cell signaling , danvers , ma ) diluted 
 at 1:200 . for detecting serum antibodies ,
the arrays were incubated 
 with serum diluted 1:2501:600 in 5% pbs milk with 0.2% tween 
 20 overnight and detected with anti - human igg - hrp ( jackson immunoresearch 
 laboratories , west grove , pa ) with tyramide ( perkinelmer , waltham , 
 ma ) .
the highly 
 immunogenic ebv - derived antigen , ebna-1 , was included as n- and c - terminal 
 fragments for positive control antigens .
registration spots for array alignment 
 were printed with purified human igg proteins . in vitro 

expression and display of target protein antigens on luminex bead 
 arrays was described in ref ( 17 ) .
briefly , each target gene was expressed as a c - terminal 
 gst - fusion protein using t7 reticulocyte lysate ( promega corporation , 
 madison , wi ) per the manufacturer s recommendations with 500 
 ng of dna .
the in vitro transcription / translation ( ivtt ) products were each captured 
 onto seromap carboxylated microspheres ( luminex corporation , austin , 
 tx ) coupled with anti - gst antisera .
protein - bound microspheres were 
 pooled together and blocked with 10% each of normal sera from mouse , 
 rabbit , goat , and rat ( jackson immunoresearch laboratories , inc . , 
 west grove , pa ) , 0.5% poly(vinyl alcohol ) ( pva , sigma - aldrich , st . 

louis , mo ) , 0.8% poly(vinylpyrrolidone ) ( pvp , sigma - aldrich , st .
louis , 
 mo ) , and 2.5% chemicon ( millipore , billerica , ma ) in pbs-1% bsa .
test 
 sera were diluted 1:80 in blocking buffer , preincubated at room temperature 
 for 1 h with rotation , and then incubated with the beads overnight 
 at 4 c while shaking .
bound igg was detected with biotin - conjugated 
 goat anti - human igg antibody ( jackson immunoresearch laboratories , 
 inc . , west grove , pa ) and streptavidin - r - pe ( molecular probes , inc .
, 
 eugene , or ) . to control for nonspecific and gst - specific autoantibody 
 background , the ratio of mfi for individual abs to the mfi for the 
 control p21-gst antigen was determined .
protein expression was confirmed 
 with a mouse anti - gst monoclonal ab ( cell signaling technology , danvers , 
 ma ) and pe - conjugated goat anti - mouse igg ( jackson immunoresearch 
 laboratories , inc . ,
ca 125 was detected by immunoassay using two monoclonal 
 antibodies ) m 11 and oc 125 , fujirebio diagnostics ) .
the upper 95th 
 percentile cutoff for healthy pre- and postmenopausal women is 35 
 u / ml . [ he4 ]
for the prescreen , 
 34 cases and 
 30 control sera ( test set , cohort 1 ) were screened on 5177 antigens 
 displayed in nappa protein array format .
each array was normalized 
 by first removing the background signal estimated by the first quartile 
 of the nonspots and then log - transforming the median - scaled raw intensities 
 to bring the data to the same scale and stabilize the variance across 
 the range of signals .
candidate antigens from the initial 5177 antigens 
 were selected if they met two different criteria : ( 1 ) comparison of 
 the 95th percentiles of the cases and controls using quantile regression and ( 2 ) comparison of the proportion of cases 
 with intensities above the 95th percentile of controls to the expected 
 number seen by chance using binomial tests , with a p - value  0.05 .
independent arrays of these 741 candidate 
 antigens were screened with a fully independent set of age - matched 
 sera consisting of 60 healthy controls and 60 patient sera .
first , we removed any duplicate antigen pairs 
 that differed by more than 3 times the median absolute deviation , 
 resulting in removal of 0.2% of spots .
second , we removed background 
 signal by subtracting the first quartile of control spot ( no dna ) 
 intensity .
third , to normalize across arrays and 384-well plates , 
 we divided the excess intensity by the median excess intensity for 
 each array and 384-well plate .
two case sera failed our qc measures 
 ( more than 20% of spots below background signal ) and were excluded 
 from further analysis .
we computed the sensitivity at an approximate 
 95% specificity for 
 each antigen as follows .
we determined a threshold by computing the 
 95% empirical percentile of the normalized intensity values of the 
 controls .
we then computed the sensitivity as the proportion of the 
 cases that exceeded that threshold and the actual specificity as the 
 proportion of the controls that did not exceed the threshold .
to identify the most sensitive antigens at a high level of specificity , 
 we used receiver operator characteristic ( roc ) curve analysis . specifically , 
 we tested the hypothesis that the partial area under the roc curve 
 ( pauc ) in the region where the specificity > 95% exceeds 0.00125 , 
 which 
 is the pauc for a noninformative diagnostic test . p - values were computed using a normal approximation to the bootstrap 
 sampling distribution , and q - values
were computed 
 using the q - value package in r. we used the training set to identify 13 potential antigen 
 biomarkers with q - values < 0.15 ( i.e. , significant 
 with 15% false discovery rate control ) .
each of these antigens had 
 a p - value < 0.01 ; for technical reasons , 12 were 
 used for further analysis .
differences 
 between cases and controls were assessed by chi - square tests . to assess 
 the value of aab to discriminate cases from healthy controls , we constructed 
 roc curves and calculated their area under the curve . associations 
 between clinical characteristics and aab detection among cases
statistical analyses 
 were performed using spss 14.0 software ( spss inc . , chicago , il ) and 
 sas ( sas institute inc . ,
our primary goal was 
 to identify serum aab biomarkers that would distinguish serous ovarian 
 cancer from healthy controls in order to guide further imaging and 
 monitoring decisions . in order to identify a biomarker panel of aab 
 in ovarian cancer from 5177 candidate antigens ,
all training and validation case and control sera were gender- and 
 age - matched and collected prior to surgery ( for cases ) under standardized 
 collection protocols .
control sera and questionnaires were collected 
 from healthy women in the boston area with no history of cancer .
table 1 shows the age distribution , menopausal status , and sample 
 collection details of the cases and controls selected for these studies . 

cases and controls did not differ in age , race , menopausal status , 
 year of blood collection , or length of storage .
only the non - serous 
 cases had a higher frequency of oral contraceptive ( oc ) use ( p = 0.003 ) compared to that of their matched controls .
as 
 expected , they also had lower mean ca 125 levels ( 85 u / ml ) compared 
 to that of serous cases .
initial screening 
 was performed using arrays expressing 5177 unique full - length cdnas 
 and case / control sera ( cohort 1 ) .
secondary screening was performed 
 using arrays expressing 741 unique full - length cdnas ( cohort 2 ) , and 
 12 antigens were retested by luminex elisa .
the specificity of the 
 top 7 antigens were determined using sera from non - serous cases / controls 
 ( cohort 3 ) and serous cases with low ca 125 levels .
includes two cases 
 that were excluded 
 from the nappa analysis due to background .
non - serous cases are 10 mucinous , 
 10 endometrioid , and 10 clear cell cases . in stage 1 of the biomarker selection
, 5177 antigens 
 were prescreened 
 with sera ( cohort 1 ) from 34 cases of serous ovarian cancer and 30 
 matched controls . for stage 2 ,
741 selected antigens were screened 
 with sera from 60 cases and 60 matched controls ( cohort 2 ) .
twelve 
 selected antigens were displayed on bead arrays and rescreened with 
 the training set to establish the performance of the assay .
finally , 
 7 promising antigens were screened by bead arrays using non - serous 
 cases ( n = 30 ) and healthy controls ( n = 30 ) , as well as sera with low ca 125 levels ( n = 20 ) and healthy matched controls ( n = 15 ) .
the 
 clinical characteristics of the serous cases in the test , training , 
 and validation sets were similar , but the ca 125 levels were lower 
 ( mean = 647 ) , on average , in the validation set than that in the test 
 set ( mean = 1232 ) .
high - density nappa protein microarrays were generated 
 for these studies for biomarker detection as described .
the 5177 individual cdnas used on these arrays 
 were derived from the arizona state university biodesign institute 
 dna repository .
these cdnas were all sequence - verified , full - length , 
 wild - type genes fused in frame with either a c - terminal gst tag or 
 an n - terminal flag tag in a vector optimized for mammalian protein 
 expression .
the content of these arrays have been described and include the breast cancer 1000 gene set , selected for their association with breast cancer 
 using bioinformatics and data mining tools .
additional genes included 
 over 300 g - coupled protein receptors ( gpcrs ) , 500 kinases , and 700 
 transcription factors .
the cdnas were co - printed on glass slides with 
 anti - tag antibodies at a high density ( up to 2300 antigens / slide ; 
 3 slides / gene set ) .
proteins were expressed and captured in situ on 
 the arrays using a coupled in vitro transcription 
dna content was confirmed 
 by picogreen , and protein expression was confirmed by probing the 
 arrays with anti - gst antibodies ( not shown ) .
the goal 
 for the first stage was to limit the number of screened antigens by 
 eliminating all of the uninformative antigens ( e.g. , no difference 
 between case and control ) .
this has the advantages of reducing the 
 false positive rate and the cost of the screen .
thus , 34 cases/30 
 control sera were screened with sera at 1:250 to 1:600 dilution on 
 5177 single antigens , and the arrays were normalized for background 
 intensity ( see statistical analysis ) .
protein 
 expression of individual spots on the microarrays was confirmed with 
 anti - gst ( not shown ) because the spotted cdnas encode c - terminal gst 
 fusion proteins .
the sera were added to the arrays , and bound igg 
 was detected with secondary antibodies .
the top 741 antigens ( supporting information table 1 ) were selected 
 if the 95th percentile of signal of cases and controls was significantly 
 different ( p < 0.05 ) and if the number of cases 
 with signals above the 95th percentile of controls was larger than 
 the number expected due to random chance ( p < 
 0.05 ) .
the goal of the second stage was to identify candidate 
 aab for further validation .
these arrays were screened with a separate 
 training set of sera from serous ovarian cancer ( n = 58 ) and sera from healthy controls ( n = 60 ) . 

two additional patient sera were removed from analysis due to unusually 
 elevated background on the arrays ( > 5 mean ) . from these data , 

12 antigens were selected as potential biomarkers for further analysis 
 based on a statistical test of the partial area under the receiver 
 operator characteristic curve ( see statistical 
 analysis ) .
the selected antigens had p < 
 0.01 and were significant with a 15% false discovery rate 
 ( fdr ) . for high - throughput screening of larger numbers 
 of sera
, the 12-antigen panel was displayed on a custom luminex microbead 
 array , which allows for rapid , multiplexed screening of sera in a 
 readily exportable , preclinical format .
first , the performance characteristics 
 of the 12 target antigens were evaluated using the same set of sera 
 ( cohort 2 ) used to screen the 741 antigens ( n = 60 
 cases/60 controls , table 2 ) .
the sensitivities 
 for these antigens are shown , using a cutoff that achieves 95% specificity . 

auc and scaled partial auc ( spauc ) values and their respective p - values are also shown .
overall , these data show that the 
 sensitivity of each individual antigen is low ( ranging from 0 to 31.7% ) , 
 with auc levels > 60% for p53 , ptgfr , ptpra ( p < 
 0.01 ) . furthermore ,
for these three antigens , the partial area under 
 the roc curve is more than 5 times greater than that for a nondiscriminating 
 test ( spauc > 5 ; p < 0.01 ) . to determine the performance characteristics 
 of the biomarker panel 
 for non - serous ovarian cancer , the luminex bead array elisa was used 
 to determine aab levels in cases derived from 10 patients with endometrioid 
 cancer , 10 patients with clear cell carcinoma , and 10 patients with 
 mucinous carcinoma ( table 3 ) . for this analysis , 

the top 3 potential antigens ( p53 , ptgfr , and ptpra ) were selected , 
 as well as 4 additional potential antigens from the validation assay . 

as expected , p53-aab were also detected in non - serous ovarian cancer , 
 but at a lower auc ( 57.4% ) , consistent with the lower frequency of 
 p53 mutations ( which are strongly associated with aab formation ) in 
 these tumors .
in contrast , aab to ptgfr 
 and ptpra were not detected in non - serous ovarian cancers .
data on 
 major risk factors for ovarian cancer ( parity , ovulatory cycles , breastfeeding ) 
 as well as levels of the biomarker ca15.3 was available on a limited 
 number of subjects in this study .
no notable correlations were observed 
 between the markers in tables 2 and 3 and the epidemiologic factors ( data not shown ) . in the training and validation cohorts ,
ca 
 125 is elevated in over 
 95% of cases due to selection of patients with serous carcinomas undergoing 
 surgery . to determine if the aab panel has potential additive benefit 
 beyond ca 125 for the detection of serous carcinomas , 20 sera were 
 identified from patients with serous carcinoma who had low ca 125 
 levels ( median 40 , range 1576.7 ) .
these cases were matched 
 by age and stage with 15 sera with high ca 125 levels ( median 2116 , 
 range 71823  010 ) .
aab 
 to at least one of the top 3 antigens ( p53 , ptgfr , and ptpra ) were 
 detected in 7/20 sera ( 35% ) in the low ca 125 cohort ( 6 by p53 alone 
 and 1 by both ptgfr and ptpra ) and no controls using cutoff values 
 of mean + 3 sd of the controls .
of the 7 sera with aab in the low 
 ca 125 cohort , 2 had stage i / ii and 5 had late stage iii / iv serous 
 carcinoma , with a median ca 125 level median of 39 ( range 1962 ) .
one challenge with the ca 125 biomarker as a screening 
 tool is false elevation in the setting of benign ovarian disease . 

we evaluated the individual performances of p53 , ptgfr , and ptpra 
 aab in a separate set of sera from 30 serous ovarian cancer patients 
 and 30 age- and gender - matched women with benign ovarian disease ( 19 
 patients with serous cytadenomas and 11 patients with mucinous cystadenomas ) .
the sensitivity of detection of aab to p53 in 
 cases was 53.3% ( auc 0.86 ) at 93.3% specificity .
the sensitivity of 
 ptgfr was 16.7% ( auc = 0.57 ) and ptpra was 13.3% ( auc 0.61 ) at 93.3% 
 specificity .
we examined the utility of these 3 aab biomarkers 
 as a diagnostic 
 panel from the combined training and validation sets .
twenty seven 
 out of 60 cases ( 45% sensitivity ) scored high ( 95% specificity threshold 
 for each antigen ) on at least one of the 3 antigens , compared to only 
 8 out of the 60 controls ( 86.7% specificity ) . however , due to the 
 rarity of ovarian cancer in the general population and the clinical 
 consequences of a false - positive result , the target specificity of 
 biomarkers for ovarian cancer is 95%99% . at 95% specificity , 

the individual sensitivities of aab to p53 was 21.7% ( auc = 0.6475 ) , 
 ptgfr was 21.7% ( auc = 0.6522 ) , and ptpra was 31.7% ( auc = 0.6525 ) . 

if at least two aab of the three were positive above the 95% specificity 
 cutoffs , then the sensitivity was 23.3% , with an improvement in overall 
 specificity at 98.3% .
using custom protein 
 microarrays , we have identified a panel of 
 12 aab biomarkers that were detected in the sera of serous ovarian 
 cancer patients at the time of clinical diagnosis of invasive cancer 
 but not in healthy women .
these individual biomarkers had sensitivities 
 ranging from 13 to 22% with specificities > 93% .
three of these 
 biomarkers , 
 p53 , ptgfr , and ptpra , were consistently selective for serous ovarian 
 cancer with individual aucs ranging from 64.8 to 73.8% across two 
 independent serum screenings and two technologic platforms ( slide 
 microarrays and bead arrays ) , involving a total of 94 cases/90 control 
 samples .
if at least two aab of the three were positive , then the 
 sensitivity was 23.3% at 98.3% specificity .
while the clinical sensitivity 
 is modest , the reproducibility , signal intensity , and clinical specificity 
 across multiple sample sets may provide utility beyond the biomarkers 
 ca 125 and he4 .
these biomarkers maintain sensitivity in the setting 
 of false - negative ca 125 levels and , unlike ca 125 , maintain specificity 
 when compared to benign ovarian disease .
this study is the first 
 demonstration of the use of programmable 
 protein microarrays for the proteomic detection of novel aab biomarkers 
 for ovarian cancer .
almost all of the sera used for this study were 
 from patients with stage iii / iv ovarian cancer ; evaluation of the 
 performance characteristics of these biomarkers will require testing 
 of prediagnostic , prospectively collected cohorts such as the roca 
 or plco trials .
it is reassuring that of the 5177 antigens we screened 
 one of the top 12 aab biomarkers was the well - established p53-aab .
none of the 12 aabs were detected in a similar 
 screen for primarily er+ breast cancer aab , although p53-aab have been detected in er - breast cancers , which 
 are more commonly mutated in tp53 .
the top antigen biomarkers did not correlate with known 
 epidemiologic risk factors , such as parity , breastfeeding , or ovulatory 
 years .
many of the top 12 antigen biomarkers that we identified have 
 also been described as being important in ovarian cancer tumor biology 
 and pathogenesis ( table 4 ) .
in addition to p53 , which we had 
 previously described , we consistently 
 identified two novel ovarian autoantigens , ptgfr and ptpra .
ptgfr 
 ( fp ) is the cell surface prostaglandin f receptor that functions to 
 initiate luteolysis in the corpus luteum .
it is aberrantly expressed 
 in endometrial carcinoma , and stimulation 
 of the receptor triggers mapk signaling and cell proliferation .
ptgfr is strongly expressed in murine ovarian 
 follicles as well as lncap prostate cancer 
 cells upon disease progression .
ptpra 
 is a cell surface protein tyrosine phosphatase that is overexpressed 
 in gastric cancers and prostate cancers and mediates signaling through erk2 .
ptpra is 
 also upregulated in the setting of her2 inhibition in breast cancer 
 cell lines . of the other candidate 
 aab biomarkers ,
validation testing using 
 our bead - array elisa and independent sera sets failed to confirm significant 
 selectivity of the biomarkers .
this may reflect poor overall performance 
 characteristics of these biomarkers or decreased sensitivity of the 
 bead arrays for the detection of aab compared to that of slide - based 
 microarrays .
dihydrofolate 
 reductase ( dhfr ) is a folate metabolism enzyme and is critical for 
 dna biosynthesis .
dhfr has long been a target for chemotherapy in 
 multiple cancers , and gene amplification 
 has been described in ovarian cancer . in our data ,
elevated serum prolactin ( prl ) 
 has been identified in the serum of ovarian cancer patients .
many proteomics - based technologies have been used 
 for the detection 
 of antigen - specific antibodies in ovarian cancer ( reviewed in ref ( 33 ) ) .
the snyder laboratory 
 used serum screening of spotted protein microarrays to identify 94 
 autoantigens .
although the difference in detection between cases and 
 controls did not reach statistical significance , they found that lamin 
 a and ralbp1 were overexpressed in ovarian cancer tissue .
lc  ms - based approaches can identify 
 native epitopes and post - translationally modified antigens ; in one 
 study , 100 aab were identified in at least one patient .
reverse - phase protein microarray can identify ptm - specific abs , and the s100a7 
 antigen has been identified .
phage - displayed 
 antigen microarrays have been used to identify 62 different antigens , 
 including rcas1 , nibrin , and rpl4 .
finally , 
 o - glycopeptide epitopes have been identified within muc1 using glycoprofiling 
 elisa assays .
additional proteomic 
 methods have been used to identify ovarian 
 cancer autoantigens . using ascites fluid and protoarrays ,
using commercial 
 protein arrays , 202 candidate ovarian antigens were identified , with dhfr being identified in at least one patient . using 2d immunoblots of exosomes ,
ovarian cancer 
 antigens plap , survivin , ny - eso-1 , grp78 , and ca 125 were identified . of the other 15 aabs that we identified ,
only 
 casein kinase 1 a has similarity to a previously identified autoantigen , 
 ck1-epsilon . in multiplexed analysis 
 of select antigens ( survivin , p53 , p16 , and cyclins b1 , d1 , a , and 
 e )
to our knowledge , other than p53 , aabs to these 
 antigens have not been identified in ovarian cancer . in summary , these 
 studies identify a potential panel of three autoantibody biomarkers 
 for the early detection of serous ovarian cancer using custom protein 
 microarrays populated with cancer - related target antigens .
 sera 
 from patients with ovarian cancer contain autoantibodies ( aab ) 
 to tumor - derived proteins that are potential biomarkers for early 
 detection . to detect aab , we probed high - density programmable protein 
 microarrays ( nappa ) expressing 5177 candidate tumor antigens with 
 sera from patients with serous ovarian cancer ( n = 
 34 cases/30 controls ) and measured bound igg . of these 
 , 741 antigens 
 were selected and probed with an independent set of ovarian cancer 
 sera ( n = 60 cases/60 controls ) . 
 twelve potential 
 autoantigens were identified with sensitivities ranging from 13 to 
 22% at > 93% specificity . 
 these were retested using a luminex bead 
 array using 60 cases and 60 controls , with sensitivities ranging from 
 0 to 31.7% at 95% specificity . 
 three aab ( p53 , ptpra , and ptgfr ) had 
 area under the curve ( auc ) levels > 60% ( p 
 < 
 0.01 ) , 
 with the partial auc ( spauc ) over 5 times greater than for a nondiscriminating 
 test ( p < 0.01 ) . using a panel of the top three 
 aab ( p53 , ptpra , and ptgfr ) , if at least two aab were positive , then 
 the sensitivity was 23.3% at 98.3% specificity . 
 aab to at least one 
 of these top three antigens were also detected in 7/20 sera ( 35% ) 
 of patients with low ca 125 levels and 0/15 controls . 
 aab to p53 , 
 ptpra , and ptgfr are potential biomarkers for the early detection 
 of ovarian cancer . 
Introduction
Methods
Results
Discussion