cancer is the second leading cause of death worldwide , accounting for about 600,000 deaths in the united states in 2012 . despite significant improvements in treatment , early detection remains the most important prognostic factor predicting of better outcome [ 24 ] . current cancer screening methods , including mammography for breast cancer , colonoscopy for colon cancer , computed tomography for lung cancer , prostate - specific antigen for prostate cancer , and papanicolaou stains for cervical cancer , have demonstrated some limitations in terms of sensitivity , specificity , complexity , cost , and compliance . serum tumor - associated antigens ( taas ) have been extensively studied for early cancer detection because of the simplicity and reliability of the tests used for their determination , such as western blot and enzyme - linked immunosorbent assay ( elisa ) . unfortunately , they are transiently secreted and rapidly eliminated from blood circulation [ 6 , 7 ] and usually reach a detectable concentration only in advanced stage of the disease . along with taas , autoantibodies are frequently detected in sera from patients affected by different types of neoplasms . this finding has been interpreted as an attempt of the immune system to block invasion and spreading of cancer cells in the organism . circulating autoantibodies have biological and biochemical characteristics that render them particularly suitable to screen subjects at cancer risk . in fact , they may develop early in the process of tumorigenesis , when premalignant or malignant cells begin to express altered molecules as a result of cell transformation [ 10 , 11 ] . in addition , they can easily be detected in the serum because of the usual high concentration and long - time stability . for these reasons , great efforts have been made in recent years to identify circulating autoantibodies directed against cancer - related proteins in order to build up tests for the early detection of neoplastic disease [ 1215 ] . in this study , we investigated the production of autoantibodies against lectin galactoside - binding soluble 3 binding protein ( lgals3bp ) in patients affected by different types of cancer . lgals3bp , also known as 90k or mac-2 bp , has been largely regarded as a taa , since it is present at elevated concentrations in the blood of cancer patients and is overexpressed in the vast majority of cancer tissues . both high serum and tumor levels of lgals3bp have been associated with a poor outcome in patients with different types of neoplasms [ 1821 ] . in fact , it can bind important molecules associated with the membrane of tumor cells , such as galectin-3 , galectin-1 , and 1-integrins [ 16 , 22 , 23 ] . additionally , lgals3bp can interact with extracellular matrix proteins such as collagen , fibronectin , and laminin [ 23 , 24 ] .   here we show that patients with different types of cancer , but not healthy subjects , develop autoantibodies against lgals3bp . this finding discloses the capability of lgals3bp to trigger a humoral immune response in cancer patients and provides the basis for further investigation on a possible use of anti - lgals3bp antibodies as biomarkers for early diagnosis of cancer . the study population consisted of 71 patients with different types of cancers , 15 gastrointestinal cancers , 13 non - small - cell lung cancers ( nsclc ) , 12 breast cancers , 10 neuroendocrine tumors ( net ) , 10 urogenital tract cancers , 7 melanomas , and 4 others ( 2 gliomas , 1 tongue cancer , and 1 osteosarcoma ) . serum was collected from total blood and stored at 20c after adding 0.01% sodium azide . human recombinant lgals3bp was immunoaffinity - purified from serum - free supernatant of human embryonic kidney ebna-293 cells ( invitrogen , carlsbad , ca , usa ) transfected with lgals3bp cdna . ninety - six well microtiter plates ( nalge nunc , denmark ) were coated with recombinant purified lgals3bp protein ( 5  g / ml in pbs ) at 4c overnight . the plate was saturated with 1% bsa and 0.05% tween-20 in pbs ( saturation buffer ) at 37c for 2  h and then incubated with 100  l of serum from healthy donors or patients at 37c for 1  h. the serum was diluted 1  :  100 in saturation buffer . after 3 washes with 0.05% tween-20 in pbs ( washing buffer ) , a second incubation was performed with 100  l of biotin - conjugated anti - human igg ( sigma , st louis , mo , usa ) , diluted 1  :  2000 in saturation buffer , at 37c for 1  h. after washing , a third incubation was performed with peroxidase - conjugated extravidin ( sigma , st louis , mo , usa ) diluted 1  :  4000 in saturation buffer , at 37c for 45  min . after washing , 100  l of tmb substrate was added to each well and the plate was shaken at room temperature for 15  min . eventually , the reaction was stopped by adding 100  l of 1  m h2so4/well and color revealed by reading absorbance at 450  nm in an automatic elisa reader . to measure serum concentration of lgals3bp a commercially available elisa kit ( diesse , siena , italy ) purified human recombinant lgals3bp ( 10  g / well ) was separated by 8% sds - page under reducing conditions and transferred to nitrocellulose using standard procedures . membrane was saturated in blocking buffer phosphate buffered saline with 0.05% tween-20 ( pbs - t ) , 5% low - fat milk , 1% bsa and at 4c overnight . after washing in pbs - t , membrane was incubated with serum from controls or patients affected by cancer , diluted in an equal volume with pbs - t , at room temperature for 1  h. after washing in pbs - t , membrane was incubated with biotinylated anti - human igg ( sigma , st louis , mo , usa ) diluted 1  :  1000 in blocking buffer at room temperature for 1  h and then with extravidin peroxidase ( sigma , st louis , mo , usa ) 1  :  500 in blocking buffer for 30  min . to identify the presence of antibodies bound to lgals3bp , the colorimetric substrate dab ( 3,3-diaminobenzidine ) each sample was assayed in triplicate and the mean value was used for statistical analyses . the normal upper cut - off value of anti - lgals3bp antibody in sera was set at the value of the mean + 2sd of the absorbance in 54 healthy donors . differences in the proportion over the cut - off limit were evaluated by fisher 's exact test . patients affected by different types of cancers showed significant increased levels of lgals3bp autoantibodies ( p < 0.001 ) compared to healthy subjects ( figure 1(a ) ) . the specific binding of antibodies to lgals3bp was confirmed in western blot , where the presence of anti - lgals3bp igg was detected as two bands at about 97  kda and 66  kda , the exact size of the protein in its full length and cleaved form , respectively ( figure 1(b ) ) . among patients , nsclc , gastrointestinal cancer , urogenital tract cancer , and net reached the highest levels of autoantibodies , while there was no significant increase in breast cancer and melanoma ( table 2 ) .   setting the normal upper cut - off limit of elisa at od 0.99 ( the mean + 2sd of the absorbance in sera from healthy individuals ) , the assay showed a sensitivity of 33% ( 26/71 patients were positive ) and a specificity of 98% ( only 1 out of 54 controls was positive ) . all cancer groups , but melanoma , showed autoantibody levels significantly above the cut - off limit ( table 2 ) . as expected , the protein was significantly higher in patients with cancer compared to normal subjects ( 13.19 versus 6.36  g / ml , p < 0.001 ) ( table 3 ) , but values did not correlate with the levels of autoantibodies ( data not shown ) . using elisa technique , we show that lgals3bp is able to elicit host immune response with igg autoantibodies production in patients affected by different types of cancer . anti - lgals3bp igg concentrations were higher in patients with nsclc , gastrointestinal cancer , urogenital tract cancer , and net than in those with breast cancer and melanoma , but the number of patients in each subgroup was insufficient to yield a statistically reliable comparison . it is generally accepted that tumor proteins perceived as nonself by the immune system and able to trigger an immune response are often overexpressed , mutated , misfolded , or endowed with posttranslational changes , such as alterations of glycosylation and phosporylation [ 28 , 29 ] . consistently , lgals3bp may evoke autoantibody production because it is overexpressed in cancer cells , and also because it may carry posttranslational alterations in its glycidic moiety . qualitative and quantitative changes in o- and n - glycosylation of proteins are frequent events in malignancies [ 30 , 31 ] and differences in the glycosylation pattern of lgals3bp have been reported in some cancer cell lines . finalistically , antitumor directed antibodies are generated in order to halt tumor initiation and progression . as this process initiates early in cancerogenesis , in a preclinical phase of the disease , autoantibodies production has been considered a useful biomarker for early cancer diagnosis [ 3235 ] . in this study , the serum levels of anti - lgals3bp igg detected in cancer patients were not correlated with those of lgals3bp , indicating that even small amount of the protein , as expected in the initial phase of cancer growth , may generate high concentrations of autoantibodies . this evidence suggests a possible role for anti - lgals3bp igg in the early detection of cancer . although it is not possible to exclude that the presence of autoantibodies might affect the correct quantification of lgals3bp by elisa , the identification of anti - lgals3bp igg in western blot indicates that the epitopes recognized by these autoantibodies are different from those recognized by the antibody used in elisa . in fact , western blot performed under reducing conditions can detect only autoantibodies directed to epitopes expressed on the primary structure of lgals3bp , while the monoclonal antibody contained in the commercially available elisa kit , known as sp2 , recognizes a conformational epitope shaped in the native form of the protein and , for this reason , is not suitable for western blotting . in cancer patients , autoantibodies are frequently directed against cellular proteins that play key roles in tumor progression , including molecules involved in cell cycle , signal transduction , proliferation , and apoptosis [ 3638 ] . as a consequence , the identification of the molecular target of autoantibodies might be of relevance in designing new antitumor agents . we can , therefore , speculate that lgals3bp could be a candidate for targeted therapies against cancer . in the past few years , the growing interest in autoantibodies as a possible tool for the early diagnosis of cancer and the identification of new targets for molecular therapy has made the development of high - throughput techniques such as serex ( serological analysis of tumor antigens by recombinant cdna expression cloning ) , phage display , protein microarray , serpa ( serological proteome analysis ) , and mapping ( multiple affinity protein profiling )   able to detect simultaneously multiple autoantibodies and their cognate taas . with these methods , several new targets have been identified , but collectively single antigens have shown low sensitivity and specificity to be used in clinical screening . to increase sensitivity , autoantibody diagnostic tests combining two or more taas [ 4143 ] or evaluating well - known biomarkers in combination with autoantibodies   have been developed . for example , a large screening study of high - risk individuals for lung cancer has validated a test measuring autoantibody levels against a panel of six taas ( p53 , ny - eso-1 , cage , gbu4 - 5 , annexin 1 , and sox2 ) . in another study , the combination of p53 autoantibodies and ca125 levels increased sensitivity for ovarian cancer from 73.8% ( ca125 ) to 85.7% ( ca 125 plus p53 autoantibodies ) . the determination of anti - lgals3bp igg presented in this study showed a very high specificity ( 98% ) , but a low sensitivity ( 33% ) , comparable to that reported for autoantibodies against single taa , ranging between 10% and 30% . therefore , our anti - lgals3bp elisa lacks sufficient sensitivity to be used in early cancer diagnosis . nevertheless , the determination of autoantibodies against lgals3bp might be useful to increase the sensitivity of tests combining multiple autoantibodies . preliminary results indicate that using a set of different autoantibodies combined with autoantibodies for lgals3bp will increase the sensitivity for breast cancer patients to 50% and maintain the high specificity ( 98% ) . these preliminary results should be tested for other types of cancers as well . in summary , our study demonstrated the presence of autoantibodies against lgals3bp in the serum of patients with different types of cancers . these autoantibodies may be used in developing screening tests for early - stage cancer detection .
purpose . 
 circulating autoantibodies have been extensively investigated as possible markers for early diagnosis of cancer . 
 the present study was carried out to investigate whether anti - lgals3bp igg autoantibodies could be classified as a biomarker for malignant tumors . 
 methods . an in - house developed enzyme - linked immunosorbent assay was used to detect autoantibodies to lgals3bp in sera from 71 patients with various types of cancers and 54 healthy subjects matched by age and gender . 
 results . 
 patients with cancer have significant higher circulating levels of anti - lgals3bp antibodies as compared to control subjects ( p < 0.001 ) . 
 the test has a sensitivity of 33% and a specificity of 98% . conclusions . 
 anti - lgals3bp igg autoantibodies are a promising biomarker for malignant tumors and could play a role in the development of a multimarker assay for the early detection of cancer .