the potential factors for primary chemoprevention of breast cancer have included the following : selective estrogen receptor modulators ( tamoxifen , raloxifene , arzoxifene , and lazofoxifene ) , aromatase inhibitors ( exemestane and anastrozole ) , isoflavones , retinoids , rexinoids , and deltanoids , polyamine synthesis inhibitors , and tyrosine kinase inhibitors ( 1 , 2 ) . all these agents have influenced the intracellular pathways of breast cancer development . as tumors of mammary gland could induce by polycyclic aromatic hydrocarbons ( 3 - 5 ) and phytoestrogens ( 6 , 7 ) , scholars have developed immunoprophylactic strategies with carcinogen - specific antibodies ( abs ) ( 8 - 11 ) . they have demonstrated that abs might protect against low doses of environmental carcinogens by decreasing systemic uptake , activation of metabolic pathways , and redistribution of carcinogens within the organism . studies on animal models with estrogen - sensitive tumors have shown that active immunization against estrogens could alter the concentration of the hormone in blood serum ( 12 , 13 ) , reduced tumor growth and increased survival time ( 14 ) . the features of antibody formation related to environmental carcinogens and sex steroids under natural conditions should determine to develop cancer immunoprevention strategies . previous studies have revealed the presence of abs against polycyclic aromatic hydrocarbons and their dna adducts in human blood serum ( 15 - 21 ) . abs to carcinogens and sex steroids in breast cancer patients ( bcp ) have only described in a few studies ( 22 - 24 ) . in the present study , abs specific to benzo(a)pyrene ( bp ) , estradiol ( es ) , and progesterone ( pg ) have evaluated to detect breast cancer risk in postmenopausal women . a total of 501 serum samples have obtained from postmenopausal women , of which 322 women have primarily diagnosed with invasive breast cancer in the regional clinical oncology hospital ( kemerovo , russia ) . each case diagnosis has confirmed morphologically and radiographically in the oncology hospital . data related to tumor size , histological type , grade , and stage have collected from surgical pathology reports . hormone receptor status ( positive or negative ) has determined immunohistochemically ( 25 ) with anti - human er antibody ( clone 1d5 ) and anti - human pr antibody ( clone pr-2c5 ) , obtained from dako corp . the following groups have identified based on the hormone receptor status : positive ( er+pr+ ) , negative ( er - pr- ) , and mixed ( er+pr- or er - pr+ ) . the median ages of participants were 61 ( ranging from 42 to 85 ) for bcp and 58 ( ranging from 39 to 80 ) for healthy women . the study protocol has conformed to the ethical guidelines of the 1975 declaration of helsinki and has approved by ethics committee of institute of human ecology sb ras ( protocol no . 12/1 ) . abs to bp , es , and pg have tested through solid - phase indirect enzyme - linked immunosorbent assays with minor modifications ( 20 ) . microtiter wells have coated with 2 g / ml bp ( es or pg ) ( sigma - aldrich , germany ) conjugated with bovine serum albumin bsa ( amresco , usa ) in 100 l phosphate - buffered saline pbs ( amresco , usa ) at room temperature overnight . the hapten - bsa conjugates have synthesized according to a previously reported method ( 21 , 26 ) . coated wells have blocked for 30 minutes with incubate buffer ( pbs with 0.05% tween 20 and 0.5% bsa ) . / well for 1 hour at 37c . bound abs have detected with goat anti - human igg antibody labeled with horseradish peroxidase ( 1/10000 dilution , sigma - aldrich , germany ) . after each assay steps wells have washed 250 l / well of pbs with 0.05% tween 20 ( amresco , usa ) . the amounts of bound abs have determined through enzymatic reaction with the chromogenic substrate tmb ( bector corp . , usa ) . the reaction has then terminated by addition of 2 n hcl and absorbance has measured at 450 nm in duplicate . the levels of abs to bp , es , and pg have expressed in arbitrary units and calculated based on the following formula : where x is bp , es , or pg ; odx - bsa is the absorbance of the binding to hapten - bsa conjugate and hapten - bsa and odbsa is the absorbance of the binding to bsa . all statistical analyses have conducted using statistica ( statsoft inc , usa , version 6.0 ) . parametric normally distributed data has statistically analyzed using shapiro - wilk s w - test , and non - parametric data have analyzed by mann - whitney u - test and  with yates correction . statistical significance has based on two - sided p values , p < 0.05 have considered statistically significant . the prognostic value of markers has assessed through roc analysis ( 27 ) , and odds ratio ( or ) has determined with 95% confidence interval ( 95% ci ) . a total of 501 serum samples have obtained from postmenopausal women , of which 322 women have primarily diagnosed with invasive breast cancer in the regional clinical oncology hospital ( kemerovo , russia ) . each case diagnosis has confirmed morphologically and radiographically in the oncology hospital . data related to tumor size , histological type , grade , and stage have collected from surgical pathology reports . hormone receptor status ( positive or negative ) has determined immunohistochemically ( 25 ) with anti - human er antibody ( clone 1d5 ) and anti - human pr antibody ( clone pr-2c5 ) , obtained from dako corp . the following groups have identified based on the hormone receptor status : positive ( er+pr+ ) , negative ( er - pr- ) , and mixed ( er+pr- or er - pr+ ) . the median ages of participants were 61 ( ranging from 42 to 85 ) for bcp and 58 ( ranging from 39 to 80 ) for healthy women . the study protocol has conformed to the ethical guidelines of the 1975 declaration of helsinki and has approved by ethics committee of institute of human ecology sb ras ( protocol no . 12/1 ) . abs to bp , es , and pg have tested through solid - phase indirect enzyme - linked immunosorbent assays with minor modifications ( 20 ) . microtiter wells have coated with 2 g / ml bp ( es or pg ) ( sigma - aldrich , germany ) conjugated with bovine serum albumin bsa ( amresco , usa ) in 100 l phosphate - buffered saline pbs ( amresco , usa ) at room temperature overnight . the hapten - bsa conjugates have synthesized according to a previously reported method ( 21 , 26 ) . coated wells have blocked for 30 minutes with incubate buffer ( pbs with 0.05% tween 20 and 0.5% bsa ) . the serum samples have diluted at 1/100 incubate buffer , and have incubated 100 l / well for 1 hour at 37c . bound abs have detected with goat anti - human igg antibody labeled with horseradish peroxidase ( 1/10000 dilution , sigma - aldrich , germany ) . after each assay steps wells have washed 250 l / well of pbs with 0.05% tween 20 ( amresco , usa ) . the amounts of bound abs have determined through enzymatic reaction with the chromogenic substrate tmb ( bector corp . , usa ) . the reaction has then terminated by addition of 2 n hcl and absorbance has measured at 450 nm in duplicate . the levels of abs to bp , es , and pg have expressed in arbitrary units and calculated based on the following formula : where x is bp , es , or pg ; odx - bsa is the absorbance of the binding to hapten - bsa conjugate and hapten - bsa and odbsa is the absorbance of the binding to bsa . all statistical analyses have conducted using statistica ( statsoft inc , usa , version 6.0 ) . parametric normally distributed data has statistically analyzed using shapiro - wilk s w - test , and non - parametric data have analyzed by mann - whitney u - test and  with yates correction . statistical significance has based on two - sided p values , p < 0.05 have considered statistically significant . the prognostic value of markers has assessed through roc analysis ( 27 ) , and odds ratio ( or ) has determined with 95% confidence interval ( 95% ci ) . the significant border levels of igg - bp , igg - es , and igg - pg between healthy women and bcp have determined through roc analysis . high abs levels ( > 4 ) were more frequent in bcp than that in healthy donors , and the difference was statistically significant ( table 1 , positions 1 and 2 ) . the ors for the bpc group were 3.5 , 4.1 , and 2.6 in relation to high abs levels against bp , es , and pg , respectively . the frequency of igg - pg ( > 4 ) in the bcp pr+ subgroup ( 39% ) was lower than that in the bcp pr- subgroup ( 50% ) , but the difference was not statistically significant . high levels of igg - pg have rarely found in the bcp er+/pr+ subgroup ( 38% ) in comparison with those in the bcp er+/pr- subgroup ( 59% ) , and the difference was statistically significant ( p < 0.05 ) . no differences have found between the other subgroups in terms of the frequency of the high levels of the three abs among the sex hormone receptors . the highest ors have found in er+/pr+ patients ( 5.5 , 5.2 , and 4.8 ) . abbreviations : bcp , breast cancer patients ; bp , benzo[a]pyrene ; ci , 95% confidence interval ; er , estrogen receptor ; es , estradiol ; igg , immunoglobulin g ; or , odds ratio ; pg , progesterone ; 95% pr , progesterone receptor . p < 0.05 between 5.2 and 5.3 . considering the possibility of different individual combinations of high and low abs levels to bp , es , and pg ( table 2 ) , the absence or low abs levels to the three haptens ( combination 1 ) have rarely found in the bcp group in comparison with that in healthy donors ( 27.9% vs. 49.7% ) . by contrast , high abs levels to the three haptens ( combination 8) have more frequently found in bcp than that in healthy women ( 36.9% vs. 5.6% ) . in this case , this value was higher than the or calculated through separate abs analysis of each hapten ( table 1 ) . abbreviations : bcp , breast cancer patients ; bp , benzo[a]pyrene ; ci , 95% confidence interval ; er , estrogen receptor ; es , estradiol ; igg , immunoglobulin g ; or , odds ratio ; pg , progesterone ; 95% pr , progesterone receptor . combinations of antibodies levels : 1 , igg - bp  4 , igg - es  4 , igg - pg  4 ; 2 , igg - bp > 4 , igg - es  4 , igg - pg  4 ; 3 , igg - bp  4 , igg - es > 4 , igg - pg  4 ; 4 , igg - bp  4 , igg - es  4 , igg - pg > 4 ; 5 , igg - bp > 4 , igg - es > 4 , igg - pg  4 ; 6 , igg - bp > 4 , igg - es  4 , igg - pg > 4 ; 7 , igg - bp  4 , igg - es > 4 , igg - pg > 4 ; 8 , igg - bp > 4 , igg - es > 4 , igg - pg > 4 . high levels of igg - bp or ig - es only without abs to other haptens ( combinations 2 and 3 ) presented similar frequency between bcp and healthy women . high levels of igg - pg only in the absence of igg - bp and igg - es ( combination 4 ) have rarely found in bcp in comparison with that , among healthy women ( 1.6% vs. 5.6% ) , but the difference was not statistically significant . combination 5 ( high levels of both igg - bp and igg - es without igg - pg ) has more frequently found in bcp than that in healthy women ( 11.5% vs. 5.6% ) , and the difference was statistically significant and the or increased to 3.8 . by contrast , combinations 6 and 7 ( high levels of igg - pg with igg - bp or with ig - es ) have rarely found in bcp compared with that in healthy women and the or approached 0.7 . the frequency of high levels of igg - bp and igg - pg without igg - es ( combination 6 ) was lower than those of igg - bp and igg - es without igg - pg ( combination 5 ) in the bcp group ( 2.5% vs. 11.8% , p = 0.009 ) . the increased ors ( 3.8 ) at high levels of igg - bp and igg - es ( combination 5 ) were statistically significant in all bcp subgroups allowing for sex hormone receptors without any differences between them . there were no any differences between healthy women and bcp as well as between subgroups of bcp when high levels of igg - pg have combined with igg - bp and with igg - es ( combinations 6 and 7 ) . the quantity and frequency of abs combinations in bcp suitable for sex hormone receptor combinations ( er / pr ) have shown in table 3 . the results have determined that the minimum or ( 0.2 ) at low levels of abs to the three haptens ( combination 1 ) and maximum or ( 26.7 ) at high levels of abs to the three haptens ( combination 8) have found in bcp er+/pr- . the statistically significant high ors ( 2.4 ) at high levels of igg - bp with igg - es ( combination 5 ) have found in all three subgroups of bcp . the statistically significant low or ( 0.02 ) at high levels of igg - bp with igg - pg ( combination 6 ) have found in bcp er+/pr+ only . abbreviations : bcp , breast cancer patients ; bp , benzo[a]pyrene ; ci , 95% confidence interval ; er , estrogen receptor ; es , estradiol ; igg , immunoglobulin g ; or , odds ratio ; pg , progesterone ; 95% pr , progesterone receptor . combinations of antibodies levels : 1 , igg - bp  4 , igg - es  4 , igg - pg  4 ; 2 , igg - bp > 4 , igg - es  4 , igg - pg  4 ; 3 , igg - bp  4 , igg - es > 4 igg - pg  4 ; 4 , igg - bp  4 , igg - es  4 , igg - pg > 4 ; 5 , igg - bp > 4 , igg - es > 4 , igg - pg  4 ; 6 , igg - bp > 4 , igg - es  4 , igg - pg > 4 ; 7 , igg - bp  4 , igg - es > 4 , igg - pg > 4 ; 8 , igg - bp > 4 , igg - es > 4 , igg - pg > 4 . the modern system for clinical prevention of bc ( 1 , 2 ) has required the use of laboratory methods to determine bc risk ; such methods included analysis of steroid - binding globulins in the blood serum ( 28 ) . cancer immunoprevention strategies have developed by some scholars which have based on induction of abs specific to environmental carcinogens ( 8 - 11 ) . however , induction of abs against estradiol reduced the growth of estrogen - sensitive tumors ( 14 ) . thus , analysis of abs against environmental carcinogens ( including phytoestrogens ) and endogenous steroid hormones could be used to determine cancer risks for the humans and develop cancer immunoprevention strategies . in this study , the risks of postmenopausal bc have calculated using immunoanalysis of abs to bp , es , and pg . the results have revealed that high levels of igg - bp only or ipg - es only have rarely found ( 6% to 10 % ) in bcp and in healthy women . combination of the high levels of igg - bp and igg - es without ipg - pg was more frequent in bcp than that in healthy women , and the or increased to 3.8.combination of the high levels of igg - pg with high levels of both igg - bp and igg - es were significantly more frequent in bcp ( 36.9% ) than that in healthy women ( 5.6% ) , and the or increased to 11.7 . previous studies have shown a negative association or absence of strong association between sex - hormone binding globulin and bc risk ( 28 ) . in a large study that have analyzed data from nine prospective trials involving women who develop or not develop bc , the risk of bc statistically significantly have increased with increasing concentrations of all sex hormones examined , whereas sex - hormone binding globulin has associated with decreased bc risk . in a case - controlled study of the european prospective investigation into cancer and nutrition , sex - hormone binding globulin levels in postmenopausal women who have developed bc have confirmed to be significantly lower than those in the control ( 29 ) . future studies should further investigate why abs to es and pg , as the sex - hormone binding immunoglobulins , with abs to bp have shown a strong positive association with bc risk . interrelations of abs to environmental carcinogens with steroid hormones and steroid hormone receptors should also be investigated . although some scholars have speculated that mucosal and serum abs reduce carcinogenesis by preventing high local carcinogen concentration ( 11 ) , other scientists have revealed that the mechanism could be induction of mucosal , not systemic , immune reaction in environmental carcinogens ( 10 ) . a previous study has also shown that active immunization using bp conjugated to protein has significantly increased the titers of bp abs and tumor formation when mice have treated with bp ( 30 ) . this study has confirmed that high levels of serum ig - bp with ig - es and ig - pg corresponding to high bc risk . in this case , induction of systemic immune reactions against environmental carcinogens could stimulate abs - formation against steroid hormones and breast carcinogenesis . however , the molecular and cellular effects of abs specific to environmental carcinogens and endogenous steroids on the mammary gland , have remained unknown .
backgroundantibodies might protect against low doses of environmental carcinogens by decreasing systemic uptake , activation of metabolic pathways , and redistribution of carcinogens within the organism . 
 the features of antibody formation in relation to environmental carcinogens and sex steroids under natural conditions should be determined to identify breast cancer risk , then to develop cancer immune prevention strategies.objectivesthe purpose of this study was to investigate antibodies specifications to benzo(a)pyrene , estradiol and progesterone in postmenopausal women with invasive breast cancer.patients and methodsa semi - quantitative non - competitive immunoassay of igg antibodies to benzo(a)pyrene ( igg - bp ) , estradiol ( igg - es ) , and progesterone ( igg - pg ) has conducted . 
 the assay has performed on 322 serum samples from patients with breast cancer and 179 serum samples from healthy postmenopausal women by using low - molecular - weight bp , es , and pg conjugated with bovine serum albumin . 
 roc analysis has also conducted to determine the odds ratio ( or).resultscombination of the high levels of igg - bp and igg - es without ipg - pg was more frequent in breast cancer patients than that in healthy women , and the or has increased to 3.8 . 
 combination of the high levels of igg - pg with high levels of both igg - bp and igg - es were significantly more frequent in breast cancer patients ( 36.9% ) than that in healthy women ( 5.6% ) , and the or increased to 11.7 . 
 these differences have peculiarly expressed in breast cancer patients with hormone status er+/pr- ( or = 26.7 ) . 
 the minimum or ( 0.4 ) has obtained at low levels of the three antibodies.conclusionsimmunoassay of antibodies against environmental carcinogens and sex steroid hormones could use to detect breast cancer risk . 
 induction of antibodies against bp for cancer immunoprevention could lead to antibody formation against steroid hormones , thereby increasing breast cancer risk .