Detection of prostate-specific antigen in breast tumors

This invention relates to the detection of prostate-specific antigen (PSA) subfractions in serum as a prognostic or predictive indicator for breast carcinoma. In particular this invention relates to an in vitro blood test for the diagnosis of breast cancer using serum PSA subfractions. Serum PSA subfractions are remarkably different in the serum from breast cancer patients, normal male patients and female patients treated for breast cancer.

FIELD OF THE INVENTION 
This invention relates to a method for the diagnosis of breast cancer using 
serum PSA subfractions. PSA serum subfractions are remarkably different in 
breast cancer patients as compared with male patients, normal females and 
females treated for breast cancer. 
BACKGROUND OF THE INVENTION 
Considerable research and related diagnosis has been undertaken in this 
field of healthcare. In order to facilitate reference to prior art 
developments and procedures, journal articles are listed at the end of 
this specification and are hereinafter referenced by number. 
Breast cancer is a leading cause of mortality and morbidity among women 
(1-4). One of the priorities in breast cancer research is the discovery of 
new biochemical markers which could be used for diagnosis, prognosis and 
monitoring (4, 5). Breast cancer is one of a few cancers that is dependent 
on steroid hormones and their receptors. Currently, estrogen and 
progesterone receptor analysis is performed routinely as an aid in 
prognosis and selection of therapy (4-6). 
Current indicators for monitoring breast tumors include: tumor size, 
estrogen receptors, progesterone receptors, age, aneuploidy, mitotic 
activity and Ki67 (29). The prognostic usefulness of these factors depends 
on their ability to evaluate which patients with breast cancer require 
aggressive adjuvant therapeutic treatment post surgery and which patients 
should be monitored. 
Mutation of the p53 tumor suppressor gene is one of the most commonly known 
genetic defects in human cancer, including breast cancer and results in 
mutant protein accumulating to high concentrations. Overexpression of p53 
protein has been found to be an independent predictor of early disease 
recurrence (29). The accumulation of p53 protein has been found to be an 
independent marker of shortened survival (30). The majority of tumors that 
do not produce mutant p53 protein are estrogen and/or progesterone 
receptor-positive (14). 
Prostate cancer is a leading cause of mortality and morbidity among men (7, 
8). Prostate tissue and cancer is also dependent on steroid hormones and 
therapy that takes advantage of this is currently routinely used (9-10). 
One of the hallmarks of prostate cancer is the appearance in serum, at 
elevated concentrations, of a 30-33-KDa glycoprotein, prostate specific 
antigen (PSA) (11). PSA is a serine protease found at high levels in 
seminal fluid and prostate epithelial cells (38). PSA production in the 
prostate is regulated by androgenic steroids, which bind to androgen 
receptors and up-regulate transcription of the PSA gene (11, 38). 
Currently PSA is a highly valuable marker for prostate cancer screening 
diagnosis, and post-surgical monitoring of prostate cancer patients, as 
well as for the detection of micrometastases (38). Normal male serum PSA 
levels are usually below 4 .mu.g/L (11,38) and it is detectable in two 
molecular forms for both normal and prostate cancer subjects; as free PSA 
or as complexed with a proteinase inhibitor, ACT (.alpha..sub.1 
-antichymotrypsin). 
Previous immunohistochemical studies found no PSA immunoreactivity in 
breast or other tumors (17) or found occasional PSA immunoreactivity with 
polyclonal but not monoclonal antibodies, suggesting cross-reactivity 
effects (18). We have now discovered the presence of PSA in breast tumors. 
Prior studies have also shown that PSA is undetectable in the serum of 
most women. A few women do have traces of serum PSA which are thought to 
be produced in the periurethral glands. In a recent study involving 1161 
normal female sera we have reported that &lt;5% of the samples had PSA 
concentrations &gt;50 ng/L (50). A recent report studying associations 
between total serum PSA levels from normal women, women with breast cancer 
and breast tumor PSA levels, indicated that there was no diagnostic or 
monitoring value of female serum total PSA (52). We have found that PSA is 
present in two subfractions in female serum. We have now discovered that 
the differences in serum PSA subfractions between breast cancer patients 
and normal women can be used to diagnose breast cancer. 
SUMMARY OF THE INVENTION 
We have discovered that serum PSA subfractions can be correlated with the 
presence of breast cancers in females. This allows for a new non-invasive 
method for the diagnosis of breast cancer which comprises a simple blood 
test to determine serum PSA subfractions for quantitation and evaluation. 
According to an aspect of the present invention is an in vitro biological 
assay for the detection of free PSA in female serum indicating the 
presence or absence of breast cancer. 
According to another aspect of the invention is an in vitro biological 
assay for the diagnosis of breast cancer in a patient comprising the 
determination of the relative mounts of free PSA and PSA-ACT complexes 
which are indicative of the presence or absence of breast cancer. 
According to an aspect of the invention is an in vitro method for the 
diagnosis of breast cancer comprising 
i) performing a highly sensitive separation technique on a serum sample to 
establish PSA subfractions; and 
ii) performing a highly sensitive assay on the PSA subfractions which is 
capable of detecting at least 1 ng/L of PSA to determine the predominant 
molecular form of PSA; and 
iii) determining the mount of PSA-ACT complex compared to free PSA to 
indicate the presence or absence of breast cancer. 
According to another aspect of the present invention is an in vitro method 
for the diagnosis of an endocrine cancer in a patient comprising, 
i) performing a highly sensitive separation technique on a serum sample to 
establish PSA subfractions; and 
ii) performing a highly sensitive assay on the PSA subfractions which is 
capable of detecting at least 1 ng/L of PSA to determine the predominant 
molecular form of PSA; and 
iii) determining the mount of PSA-ACT complex compared to free PSA to 
indicate the presence or absence of breast cancer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
PSA in Breast Tumour Extracts 
We have carried out extensive investigations on breast tumors and 
surprisingly, found that twenty-nine percent of the breast tumor extracts 
were found positive for PSA (cutoff level 0.05 .mu.g/L or 0.03 ng/mg total 
protein). PSA was associated with tumors that were estrogen and/or 
progesterone receptor-positive (P&lt;0.002). No association was found between 
PSA levels and levels of the p53 tumor suppressor gene product (P=0.37). 
High performance liquid chromatography revealed that PSA is present in the 
tumor predominantly in its free, 30-33 KDa form. PSA-positive tumors were 
associated with younger (premenopausal) women (P=0.012) and earlier 
disease stage (P=0.064). It appears that PSA production is induced by 
steroid hormone receptor-ligand complexes. 
The cutoff value of 0.05 .mu.g/L (0.03 ng/mg total protein) for PSA in the 
breast cytosols was arbitrarily selected based on the PSA assay 
sensitivity. PSA values &gt;0.05 .mu.g/L can be easily and precisely 
quantified by using the developed assay of the invention. It is 
appreciated that various assay techniques may be used to detect PSA; for 
example, enzyme immunoassay, radioimmunoassay, chemi- or bio-luminescent 
immunoassay, fluorescent immunoassay and DNA-based assays to detect 
expression of the PSA gene at the mRNA level. 
In accordance with a preferred aspect of the invention, an assay comprising 
an ultrasensitive detection method for prostate-specific antigen in breast 
tumor extract involving time-resolved fluoroimmunoassay is provided. 
Breast tumor extract is incubated with monoclonal anti-PSA antibody. 
Biotinylated polyclonal or monoclonal antibody specific to PSA is added to 
bind to any bound PSA. Alkaline phosphatase-labelled streptavidin (SA-ALP) 
is added. The activity of ALP is measured by adding the substrate 
5-fluorosalicyl-phosphate and then adding Tb.sup.3+ -EDTA to form a 
fluorescent chelate. Fluorescence is measured over time to indicate the 
presence of PSA. The presence or absence of PSA can be used as a 
prognostic and predictive indicator of breast carcinoma. The invention's 
detection method can also be used for detecting the presence of other 
markers or substances, such as p53 protein, using the appropriate 
antibody. 
The data we have established and as summarized in Table I, establishes an 
association between breast tumors and tissue level of PSA. 525 breast 
tumor extracts were analyzed for PSA with the results as shown in Table I. 
From these tumor extracts, 374 (71.2%) had PSA levels &lt;0.05 .mu.g/L and 
were considered negative for PSA. One hundred and fifty-one (28.8%) of the 
tumor extracts had PSA levels &gt;0.05 .mu.g/L, 96 (18.3%) had PSA levels 
&gt;0.1 .mu.g/L and 49 (9.3%) had PSA levels &gt;0.3 .mu.g/L. Samples with a PSA 
concentration of &gt;0.3 .mu.g/L, which is potentially measurable by 
commercial kits, were also analyzed by the Hybritech Tandem.RTM. M-R PSA 
kit, by the IRMA-Count.RTM. PSA kit and by the Abbott IM.sub.x Kit. The 
results are shown in FIG. 1. 
To further exclude the possibility of non-specific effects, the assay was 
repeated for 25 highly positive samples (PSA &gt;0.3 .mu.g/L) under the 
following conditions: (a) the assay was run in the absence of capture 
mouse monoclonal anti-PSA antibody (b) the assay was run by using an 
irrelevant capture mouse monoclonal antibody (against alpha-fetoprotein) 
(c) the assay was run after substitution of the polyclonal rabbit 
detection antibody with biotinylated rabbit IgG. In all cases, background 
signals were obtained verifying that non-specific effects were absent. 
PSA immunoreactivity was further investigated in two breast tumor extracts 
by using high performance liquid chromatography (HPLC). One male serum 
sample with a PSA concentration of 4.27 .mu.g/L by TR-FIA and one negative 
breast tumor extract were used as positive and negative controls. Analysis 
of PSA was performed in the HPLC fractions and the results are shown in 
FIG. 2. The PSA-negative breast tumor extract, run between the positive 
samples, gave undetectable readings in all fractions, in all cases. The 
PSA immunoreactivity in the two breast tumor extracts, elutes as a single 
peak at fraction 45 and corresponds to a molecular weight of approximately 
30-33 KDa. The PSA immunoreactivity in the male serum sample elutes in two 
peaks at fractions 40 and 45 and corresponds to molecular weights of 
approximately 100 KDa and 30-33 KDa, respectively. These two peaks 
correspond to PSA bound to .alpha..sub.1 -antichymotrypsin and to free 
PSA, respectively (13, 15, 16). These findings demonstrate that the PSA in 
the breast tumor extracts is present exclusively in the free 30-33 KDa 
form. 
In order to exclude the possibility of contamination of the extracts, six 
PSA-positive and six PSA-negative breast tumors that were stored frozen at 
-70.degree. C. were reextracted. Rerun of the fresh extracts with the 
TR-FIA assay confirmed the original results in all cases. Ninety-four 
breast tumor extracts were also obtained from another steroid hormone 
receptor laboratory serving different hospitals in Toronto. From these, 17 
(18%), 12 (13%) and 5 (5.3%) had PSA values &gt;0.05, &gt;0.1 and &gt;0.3 .mu.g/L, 
respectively. 
Recovery experiments done by spiking PSA-negative tumor extracts with 
seminal plasma PSA gave values averaging 83% of the amount of exogenous 
PSA added. Dilution experiments were performed by diluting a breast tumor 
extract with a high PSA concentration (20.4 .mu.g/L) with either a 6% 
(w/v) bovine serum albumin solution or a PSA-negative breast tumor 
extract. The obtained values, at dilutions ranging from 2 to 32-fold, were 
very close to those predicted by the PSA-value in the undiluted specimen 
(100.+-.5%). A batch of 16 breast tumor extracts (four with PSA &lt;0.05 
.mu.g/L and twelve with PSA &gt;1 .mu.g/L) were also sent to two different 
laboratories performing routine PSA assays by the Hybritech and DPC 
methods. In both cases, their values were very similar to the ones 
obtained by our method. These data further demonstrate that the 
invention's PSA detection results are not due to any non-specific effects 
and that contamination is very unlikely. 
Although we describe detection of PSA with a time-resolved 
immunofluorometric technique, it is understood that those skilled in the 
art may use other techniques presently available or future immunological 
techiques for PSA quantification to at least 0.03 ng/mg of total protein. 
For example, techniques capable of such sensitivity include 
chemiluminescence with acridinium esters as labels, enzymatically 
triggered chemiluminescence with alkaline phosphatase and dioxetanes 
substrates luminol chemiluminescence enhanced by horseradish peroxidase, 
immunoassays using alkaline phosphatase and the fluorogenic substrate 
4-methylumbelliferyl phosphate or p-nitrophenyl phosphate, immunoassay 
using horseradish peroxidase and substrates like ABTS and 
tetramethylbenzidine, time-resolved immunofluorometric assays with 
Eu.sup.3+ as label and methods based on electroluminescence. 
In addition, PSA expression may also be detected by determining whether 
mRNA for PSA is present in a breast tumor sample. The preferred procedure 
for detecting mRNA for PSA is by PCR amplification. Total RNA or mRNA is 
isolated from breast tumor samples and cDNA synthesized by reverse 
transcription. PCR amplification of cDNA is accomplished using PSA 
specific primers. A probe is used to detect cDNA for PSA. Other methods 
for detecting an RNA for PSA may also be used, such as, the Northern Blot 
technique. 
For most of the tumor extract samples analyzed for PSA, data for estrogen 
(ER) and progesterone (PR) receptor concentrations was available. Also 474 
samples were analyzed for the presence of the p53 tumor suppressor gene 
product, using a method previously described (14). Tumors were then 
classified as being positive or negative for ER, PR, p53 and PSA using the 
following negativity cutoff levels: &lt;10 fmol/mg of total protein for ER 
and PR (14, 30,31); &lt;3 U/L, for p53 (equivalent to 0.02 ng/mL) (14) and 
&lt;0.05 .mu.g/L for PSA. The data are summarized in Table II. 
There is a significant association between the presence of estrogen and/or 
progesterone receptors and the presence of PSA in the tumors (P&lt;0.002). 
PSA is independently associated with ER and PR because minors which are 
either ER(+) only or PR(+) only still have higher percentage of positivity 
for PSA in comparison to minors which are negative for both receptors. 
Additionally, the highest percentage of PSA-positive tumors is associated 
with tumors that are positive for both the ER and PR (Table II). There is 
no association between the presence of PSA and the presence of the p53 
tumor suppressor gene product (P=0.37). It has recently been shown that 
the latter is strongly associated with estrogen and/or progesterone 
receptor-negative tumors (14) an association also shown in Table II for 
the samples of this study 
Correlation studies using linear regression analysis between ER and PR and 
PSA, for all samples of this study (N=525) gave the following Pearson 
correlation coefficients: r=-0.023, not significantly different from zero 
(NS), P=0.60 for ER and r=-0.015, (NS), P=0.71 for PR When only the 
PSA-positive tumors were used for correlation (N=151) the following 
Pearson correlation coefficients were obtained: r=-0.015, (NS), P=0.85 for 
ER and r=-0.068, (NS), P=0.40 for PR. 
Some breast tumors had very high PSA levels. Highest values were obtained 
for five tumors in which PSA levels were &gt;20 .mu.g/L in the extracts and 
between 200-1000 ng of PSA per g of breast tumor tissue. 
Association analysis between PSA presence in breast tumors and patient age 
gave the results shown in Table III and FIG. 3. PSA was distributed 
preferentially in younger (premenopausal) patients and this preference was 
statistically significant (P=0.012). 
Tumor stage was available in 203 patients. The results of the distribution 
of PSA-positive tumors in various stages is given in Table IV and FIG. 4. 
Clearly, there is a trend for the PSA-positive tumors to be preferentially 
associated with lower disease stage. 
PSA-positive tumors are predominantly ER(+) and PR (+). The presence of PSA 
in a tumor is indicative of functional ER and PR because PSA is closely 
associated with the PR (Table V). PR is a product of the action of the ER 
and is indicative of functional ER. Thus, monitoring PSA would be a useful 
test to identify patients who possess functional ER and PR. These patients 
are the ones most likely to respond to endocrine treatment which currently 
consists of administering one or more of the following: Antiestrogens, 
antiprogestins, antiandrogens, progestins, androgens, glucocorticoids. 
Thus, the classification of patients as PSA(+) and PSA(-) may be useful to 
select those who will benefit from endocrine treatment. 
In addition, a subgroup of PSA-positive and ER-negative patients was 
suprisingly found to have a good prognosis and respond well to endocrine 
treatment. In order to examine the prognostic significance of PSA in the 
subsets of patients who are ER-negative or ER-positive, the hazard ratio 
between PSA-positive and PSA-negative patients was calculated for two 
subsets being the ER-negative and the ER-positive groups, using the Cox 
regression model. The analysis was done at two cut-off levels of the 
receptors, 10 fmol/mg or 20 fmol/mg since with the receptor assays used, 
levels between 10-20 fmol/mg are considered equivocal. The results of the 
analysis are shown in Table 6. In the ER-positive group the risks of 
relapse were almost identical between PSA-positive and PSA-negative 
patients, which was expected since it is known that steroid hormone 
receptors are favourable prognostic indicators in breast cancer. However, 
in the ER-negative group, the risk of relapse was substantially reduced 
when the tumors were PSA-positive (hazards ratio 0.13-0.20). The 
difference was statistically significant when the cutoff level of the 
receptors was 20 fmol/mg due to the increase in the number of patients in 
this subgroup. The hazards ratio in the ER-negative subgroup remained very 
low even when nodal status, clinical stage and histological type were 
controlled in the analysis. 
The risk for cancer relapse was significantly lower in patients with 
PSA-postive tumors than in patients with PSA-negative tumors. The hazard 
ratio for relapse of PSA-positive patients and PSA-negative patients was 
0.32. A similar hazard ratio for overall survival was also observed. 
Overall and relapse-free survival curves are shown in FIG. 6. The 
probabilities of relapse-free and overall survivals were substantially 
higher in the PSA-positive patients than in the PSA-negative ones. FIG. 6 
demonstrates that PSA-positive patients relapse less frequently and live 
longer than PSA-negative patients and that this difference is statistcally 
significant (P =0.06 and 0.04, respectively). Of the 174 patients, 42 had 
cancer relapse and 27 died. The overall follow-up time for these patients 
ranged between 7 and 67 months with a median of 33 months. PSA 
immunoreactivity higher than 0.03 ng/mg was detected in 27% of the 
patients (47/174). Without considering the follow-up time PSA-positive 
patients were less likely to relapse or die than PSA-negative patients 
(11% of PSA-positive patients versus 29% of PSA-negative patients for 
cancer relapse and 6% of PSA-positive patients versus 19% of PSA negative 
patients for death). 
The data shows that breast tumors produce PSA, an antigen that was 
originally thought to be highly specific for the prostate. Previous 
immunohistochemical studies found no PSA immunoreactivity in breast or 
other tumors (17) or found occasional PSA immunoreactivity with polyclonal 
but not monoclonal antibodies, suggesting cross-reactivity effects (18). 
The percentage of tumors producing PSA is significant (approximately 29%) 
similar or higher to the percentage of tumors with amplification of the 
HER-2 oncogene (19). The PSA form in the tumor has a molecular weight of 
approximately 30 Kda and corresponds to the free PSA molecule. 
The production of PSA by breast tumors is due to PSA gene upregulation by 
steroid hormone receptors bound to either progestins, androgens or 
glucocorticoids (FIG. 5). This is indicated by the finding that most 
tumors producing PSA are steroid hormone receptor-positive. From the 151 
PSA-positive tumors, only 20 were negative for estrogen and/or 
progesterone receptors. From these, fifteen had detectable estrogen and/or 
progesterone receptor levels but their concentration was below the cutoff 
point of 10 fmol/mg of protein. Only five PSA-positive tumors (3.3%) had 
undetectable estrogen and progesterone receptor levels by the method used. 
In these five tumors the PSA immunoreactivity of the extracts was 
relatively low (0.05, 0.06, 0.14, 0.17 and 0.37 .mu.g/L). 
Recent reports suggest that PSA expression in the prostate may be under the 
direct influence of hormones, namely synthetic androgens or testosterone 
(20-23). Our observation that the presence of PSA in breast minors is 
dependent upon the presence of the steroid hormone receptors and that 
there is no correlation between levels of PSA and receptors, indicate that 
the receptors are necessary but not sufficient for PSA production. In 
addition, one or more as yet unidentified ligands interact with the 
steroid hormone receptors to form a complex that regulates PSA gene 
derepression (FIG. 5). Active ligand-receptor complexes apparently exist 
in only 32% of the steroid hormone receptor-positive tumors. It is not 
clear if in the rest of the steroid hormone receptor-positive tumors the 
ligand(s) is/are absent, the receptors are defective as previously 
suggested (24) or the ligand-receptor complexes are formed but are somehow 
ineffective at the level of gene derepression. 
This mechanism for PSA gene derepression in breast cancer is further 
supported by the finding that PSA production is associated with younger 
patient age (P=0.012, Table 3). In patients over the age of 55, only 24% 
of tumors produce PSA even if the estrogen or progesterone 
receptor-positive tumors are over 80% of the total. In patients under the 
age of 35, 33% of tumors produce PSA even if the estrogen and/or 
progesterone receptor-positive tumors are only 50% of the total. To 
further demonstrate the effect of age on PSA production the percentage of 
tumors that produce PSA from the total number of estrogen or progesterone 
receptor-positive tumors was calculated. These values are 67% (6/9) and 
75% (6/8), respectively, for the age group &lt;35 years and 29.7% (80/269) 
and 38.1% (30/210), respectively, for the age group &gt;55 years (data from 
Table 3). The higher PSA positivity rate among younger patients may be 
related to production of the putative ligands of FIG. 5 by the functioning 
ovaries. 
Although disease stage was available only for 203 patients, the association 
analysis between PSA production and disease stage demonstrates (Table IV 
and FIG. 4) that there is a dear trend for PSA-positive tumors to be 
preferentially associated with lower disease stage. The P values did not 
fall below 0.05 because of the relatively small number of samples in some 
patient groups. 
A practical implication of these findings is that the PSA gene regulation 
mechanism may be used for treatment of breast tumors. An examination of 
the ligands involved in steroid hormone receptor binding and PSA gene 
regulation in breast cancer may assist in this treatment. Breast tumors 
producing PSA constitute a sizable group (29% of patients) which may be 
examined in retrospective or prospective studies to establish if patients 
have a different prognosis or favourable response to selected therapy. 
The data indicates that PSA is a favourable prognostic indicator because it 
is associated more strongly with tumors that are positive for both 
receptors, with lower disease stage and with improved patient survival 
(FIG. 6). In the breast tumor, PSA is present in the predominantly free 
30-33 kDa form. The suggested mode of PSA production (FIG. 5) based on the 
findings that the overwhelming majority of PSA-positive tumors have 
detectable receptors (146/151 or 97%) and that younger patients are more 
positive than older patients, lead to the conclusion that the PSA-positive 
tumor is a subgroup that possesses "effective" receptors, capable of gene 
regulation, as exemplified by PSA production. Then PSA-positive tumor 
patients will be most likely to respond to steroid hormone therapy. This 
was recently suggested for the steroid hormone receptor-inducible pS.sub.2 
-BCEI protein, another potential prognostic indicator in breast cancer 
(25, 26). 
A significant proportion of breast tumors (29%) produce PSA. PSA production 
is associated with steroid hormone receptor-positive tumors, younger age 
and earlier disease stage. PSA can be used as a routine prognostic marker 
for breast carcinoma and may play a role in disease initiation and 
progression. The invention's time-resolved fluoroimmunoassay is sensitive 
enough to detect levels of PSA as low as 0.05 ug/L in breast tumor 
extracts which equivalent to approximately 0.03 ng of PSA per mg of total 
protein. 
Serum PSA Subfractions 
Previously we had suggested that total serum PSA had no diagnostic or 
monitoring value. We however, have now discovered that the free 30-33 kDa 
form of PSA is specifically related to breast cancer and that this free 
form of PSA could be quantitated in serum to provide a non-invasive in 
vitro method to diagnose breast and other cancers. 
To test this hypothesis male sera, sera from women with breast cancer, and 
sera from women with breast cancer post-operatively were studied. All 
types of serum samples with the exception of male sera, were selected on 
the basis of their total PSA level (.gtoreq.16 ng/L), and the availability 
of sufficient sample volume (&gt;100 uL) for HPLC analysis (Table 7). In 
general, they approximately represented samples from the upper pentile of 
their respective serum type. 
Separation of serum immunoreactive PSA was done by HPLC followed by 
immunofluorometric analysis of their corresponding fractions. HPLC was 
used because it is currently the most sensitive method by which to 
separate the PSA species, however it is understood that any other method 
developed for these purposes could also be used. In addition, the 
detection of the PSA present in the fractions can also be done using 
several different assays such as enzyme immunoassay, radioimmunoassay, 
chemi or bio-luminescent immunoassay and fluorogenic immunoassay. 
It is also understood by those skilled in the art that PSA subfractions can 
also be assessed using other techniques including the direct measurement 
of free PSA and/or PSA-ACT complexes using immunoassays without the need 
for HPLC or other separation of serum fractions. Such assays utilize 
specific monoclonal antibodies produced against PSA and have been 
described in the literature (16). These assays are the preferred mode for 
determining the relative mount of free and complexed PSA to indicate the 
presence or absence of breast cancer. 
The results revealed for three normal female sera, that the molecular form 
of immunoreactive PSA is the complexed form; PSA bound to ACT (PSA-ACT; 
.about.100 KDa), which peaks at fraction 30.+-.1 (FIG. 7; panels A,B,C). 
Free PSA was not detectable (see below). Immunofluorometric analysis of 
serum fractions from three preoperative females with primary breast cancer 
demonstrated that the predominant molecular form of PSA is free PSA 
(F-PSA; .about.33 KDa), which peaks at fraction 39.+-.1 (FIG. 7; panels 
D,E,F). PSA-ACT complex constitutes a minor molecular form in the 
presurgical serum of the three females with breast cancer. Fractions from 
seven postsurgical sera were also analyzed in the same manner PSA (FIG. 
8). Our results show that the predominant molecular form of PSA in the 6 
out of 7 postoperative sera, exists as a complex with ACT. The present 
clinical status for cases G and H of Table 7 is unknown, but all other 
subjects are in remission for the times indicated. The predominant 
molecular form of PSA for case G is F-PSA. 
Immunofluorometric PSA determination of serum fractions from three normal 
male sera and three sera from post-radical prostatectomized subjects with 
prostate cancer indicated that the major PSA species in all of these serum 
samples is the PSA-ACT complex. Representative data are shown in FIG. 9. 
F-PSA is the minor molecular form of PSA in these sera. 
It is known that PSA is primarily produced and secreted by the columnar 
epithelial cells of the prostate (11, 38). Briefly, PSA is translated as a 
261 amine acid preproPSA precursor. It enters the secretory pathway when 
the signal peptide represented by the pre-region (17 residues) is removed 
in the endoplasmic reticulum. The resulting inactive proPSA (zymogen) is 
exocytosed into the lumina of the prostate ducts. The release of seven 
N-terminal residues results into the 237-amine acid mature extracellular 
form, enzymatically active PSA. The protease(s) responsible for the 
formation of the active PSA via proPSA cleavage has not been identified 
yet. The primary biologic role of PSA is to increase sperm motility via 
the cleavage of the major seminal gel forming proteins semenogelin I, II, 
and fibronectin in seminal fluid (SF) into small peptides. Although the 
majority of the PSA in SF is enzymatically active, about 20-30% is 
inactive primarily due to clipping between residues 145-146 
(lysine-lysine) (49). The nicked PSA remains connected by the internal 
disulfide bonds, but does not complex to any pretense inhibitors. 
The predominant form of immunoreactive PSA in the male serum is the one 
complexed to ACT (15, 49). Our results confirm that the minor PSA species 
is indeed F-PSA in normal male serum and serum of post-radical 
prostatectomy prostate cancer patients (FIG. 9). The F-PSA in serum has 
not been fully characterized. The uncomplexed and enzymatically inactive 
PSA could be either the internally clipped PSA or the 244 amine acid 
proform (zymogen) or even KLK2, a kallikrein highly homologous to PSA. 
Although PSA may possibly be autocatalytic, the cleavage sites observed 
are highly suggestive of a trypsin like enzyme. A speculation has been 
made that this trypsin-like activity and hence the inactivation of PSA by 
nicking may be attributable to KLK2 (54). However, it seems that this 
inactivation occurs before PSA is released into the circulation, since the 
huge excess of protease inhibitors in the blood would have likely 
complexed with the otherwise non-clipped enzymatically active PSA. 
The molecular characterization of immunoreactive PSA in cytosolic breast 
tumor extracts and normal breast tissue has shown that the predominant 
molecular form is the F-PSA (40, 43). However, the presence of an 
enzymatic activity or the determination of its physicochemical and 
biomolecular properties have not been examined in breast as yet, mostly 
due to the production and presence of minute amounts in comparison to 
those of the prostate gland. We have previously demonstrated that fewer 
than 5% of women have serum PSA concentrations of .gtoreq.50 ng/L (50). A 
recent study involving the measurement of PSA with an optimized 
ultrasensitive assay (biological detection limit of 1 ng/L) (53) from sera 
of 212 normal women, revealed that 32% of the women had PSA values of 
.ltoreq.1 ng/L while the median was 2 ng/L. We have previously reported, 
in a study examining female serum total PSA levels, that there is no 
association of breast tumor PSA levels with serum PSA either pre or post 
operatively, and also no substantial difference of serum PSA levels 
between normal women and women with breast cancer (17). The results of the 
present study indicate that the predominant and quite possibly the only 
molecular form of circulating PSA existing in the serum of normal women is 
PSA complexed with ACT (FIG. 7). Moreover, the predominant molecular form 
of PSA in the pre-surgical serum of women with breast cancer is the F-PSA; 
presumably the internally clipped and non-enzymatically active form of PSA 
the proPSA molecule or KLK2. The results indicate that the female serum 
presents differences with respect to the presence of PSA molecular form 
variants between normal and breast cancer afflicted subjects. 
Determination of the PSA molecular forms in seven post-operative sera from 
women with breast cancer, indicated with one exception, which we speculate 
to be a relapsed case, that the major PSA molecular form is the PSA-ACT 
complex. The degree of post translational modification with reference to 
PSA clipping could be a distinguisable feature for the diagnosis and 
monitoring of breast cancer. 
The data presented here allow us to propose a simple diagram coveting PSA 
production by breast epithelial cells (FIG. 10). We suggest that normal 
breast epithelial cells secrete enzymatically active PSA which binds to 
.alpha..sub.1 -antichymotrypsin when it enters the general circulation. 
Breast cancer cells seem to produce enzymatically inactive PSA which does 
not bind to ACT and circulates as a free 33 KDa protein. Free PSA may 
represent internally clipped PSA, pro PSA, KLK-2 or even mutant PSA 
produced by the tumor. Alternatively, the tumor may produce an 
endopeptidase which cleaves enzymatically active PSA. The consequences of 
the loss of enzymatically active PSA from the breast are not known, nor it 
is known if this loss occurs before or after the malignant transformation. 
To summarize, we have examined the molecular forms of PSA in the serum of 
normal women and women with breast cancer. The results indicate that the 
molecular forms of PSA differ in females with or without breast cancer. 
The clinical value of PSA molecular forms was also examined by other 
investigators for males (57). Determination of the proportions of F-PSA 
and PSA-ACT may assist in the discrimination of prostate cancer and benign 
prostatic hyperplasia (BPH) as well as other endocrine cancers. The 
prospect of measuring PSA molecular forms in the female serum appears 
clinically useful for the diagnosis and management of breast cancer. 
Furthermore, measuring active and inactive free forms of PSA may also 
provide to be useful for the diagnosis of cancers. 
METHODS 
Patients--Breast Tumors 
Approximately 500 breast tumor extracts were, analyzed for steroid hormone 
receptors, for the p53 tumor suppressor gene product and for PSA, using 
the invention's new, highly sensitive immunofluorometric procedure. 
All primary tumors used in this study were collected from about ten 
different hospitals in Ontario. Primary breast tumor tissue was 
immediately stored in liquid nitrogen after surgical resection, 
transported to the laboratory and stored subsequently at -70.degree. C. 
until extraction was performed (.about.1-2 weeks). Approximately 0.5 g of 
tumor tissue was weighed out, smashed with a hammer if necessary, and 
pulverized in a Thermovac tissue pulverizer with liquid N.sub.2. The 
resulting powder was transferred into 50 mL plastic tubes along with 10 mL 
of extraction buffer (0.01 mol/L Tris, 1.5 mmol/L 
ethylenediaminetetraacetic acid, 5 mmol/L sodium molybdate, pH adjusted to 
7.40 with 5 mol/L HCl). The tissue powder was homogenized on ice with a 
single 5s burst of a Polytron homogenizer. The particulate material was 
pelleted by 1 h centrifugation at 105,000 g. The intermediate layer 
(cytosol extract) was collected without disturbing the lipid or 
particulate layers. Protein concentration of the cytosol extract was 
determined by the Lowry method and the extracts were stored at -70.degree. 
C. until analysis (up to three weeks). In determining the total protein of 
tumor tissue sample to be tested, the protein concentration of the extract 
may provide the basis for such determination. Hence, the detection level 
of 0.03 ng of PSA per mg of total protein is determinative for deciding 
PSA (+ve) or (-ve). Stability studies have revealed that the p53 protein 
and PSA in the cytosol extracts are stable for at least four months at 
-70.degree. C. 
Estrogen and Progesterone Receptors 
Quantitative analysis of estrogen and progesterone receptors (ER, PR) was 
measured using the Abbott enzyme immunoassay kits (Abbott Laboratories, 
North Chicago, Ill. 60064). The kits were used according to the 
manufacturer's instructions. 
PSA and p53 Measurement 
Analysis of PSA and p53 was performed using the invention's time-resolved 
fluoroimmunoassay. 
Instrumentation 
For measuring liquid-phase Tb.sup.3+ fluorescence in white microtiter 
wells, we used the CyberFluor 615.RTM. Immunoanalyzer, a time-resolved 
fluorometer. The time-gate settings of the instrument and the interference 
filter in the emission pathway were the same as described elsewhere 
(32,33). 
PSA MEASUREMENT 
Reagents and Solutions 
All reagents were purchased from Sigma unless otherwise stated. The coating 
solution was a 50 mmol/L Tris buffer, pH 7.80, containing 0.5 g of sodium 
azide per liter. The wash solution was a 5 mmol/L Tris buffer, pH 7.80, 
containing 0.15 mol of NaCl and 0.5 g of polyoxyethylenesorbitan 
monolaurate (Tween 20) per liter. The substrate buffer was a 0.1 mol/L 
Tris buffer, pH 9.1, containing 0.15 mol of NaCl, mmol MgCl.sub.2 and 0.5 
G of sodium azide per liter. The substrate stock solution is a 10 mmol/L 
diflunisal phosphate (DFP) solution in 0.1 mol/L NaOH. It is available 
from CyberFluor Inc., Toronto, Canada. The developing solution contains 1 
mol Tris base, 0.4 mol NaOH, 2 mmol, TbCl.sub.3 and 3 mmol of EDTA per 
liter (no pH adjustment). This solution is prepared as described 
previously (23, 24) and is commercially available by CyberFluor. The assay 
buffer is a 50 mmol/L Tris buffer, pH 7.80, containing 60 g of BSA, 0.5 
mol of KCl, 0.5 g of sodium azide, 50 mL of normal mouse serum and 5 g of 
Triton X-100 per liter. The polyclonal biotinylated detection antibody and 
SA-ALP diluent is a 50 mmol/L Tris buffer, pH 7.80, containing 60 g of BSA 
per liter. The GARlg-ALP conjugate diluent is the same as the polyclonal 
biotinylated detection antibody diluent but also contains 4% (v/v) of goat 
serum. The blocking solution was a 50 mmol/L Tris buffer, pH 7.80, 
containing 10 g of BSA per liter. 
Antibodies 
The mouse monoclonal MBP0405 and the rabbit polyclonal PBG0101 anti-PSA 
antibodies were purchased from Medix Biotech, Foster City, Calif. 94404. 
The SA-ALP conjugate was purchased from Jackson ImmunoResearch, West 
Grove, Pa. 19390. The alkaline phosphatase-conjugated affinity purified 
goat anti-rabbit IgG, Fc fragment specific (GARlg-ALP) was also purchased 
from Jackson. A poIyclonal rabbit antibody against .alpha..sub.1 
-antichymotrypsin was purchased from Dakopatts (Glostrup, Denmark). 
Standards 
Because of the unavailability of a universally accepted standard from PSA, 
for our studies we used PSA standards in a 50 mmol/L Tris buffer, pH 7.80, 
containing 6% (w/v) of BSA. A stock PSA solution, prepared from PSA 
purified from human seminal plasma, was purchased from Scripps 
Laboratories, San Diego, Calif. 92121. Our final standard solutions were 
calibrated against standards fro the Hybritech Tandem-PSA kit (Hybritech 
Inc., San Diego, Calif. 92126). For routine use we recommend six PSA 
standards with concentrations of 0, 0.025, 0.1, 0.5, 2 and 10 .mu.g/L. 
These are stable for at least one month at 4.degree. C. 
Biotinylation of the Polyclonal Anti-PSA Antibody 
The polyclonal anti-PSA antibody, purified by ion-exchange chromatography, 
was dialyzed overnight against five liters of a 0.1 mol/L sodium 
biocarbonate solution. This stock solution (.about.2 mg/ml) was diluted 
2-fold with a 0.5 mol/L carbonate buffer, pH 9.1. To this solution we 
added 1 mg of NHS-LC-Biotin (from Pierce Chemical Co., Rockford, Ill.) 
dissolved in 50 .mu.L of dimethylsulfoxide, under continuous stirring and 
incubated for 2 h at room temperature. This biotinylated antibody was used 
without further purification and stored at 4.degree. C. for at least six 
months. 
Coating of Microtiter Wells 
White, opaque 12-well microtiter polystyrene strips were obtained from 
Dynatech Laboratories, Alexandria, Va. 22314. The wells were coated 
overnight at room temperature with 500 ng/100 .mu.L/well of coating 
monoclonal anti-PSA antibody in the coating buffer. Before use, the wells 
were washed x 2 and blocked for 1 hour with 200 .mu.L/well of the blocking 
solution. 
Assay Procedure 
Wash the strips x 6. In each well pipet 50 .mu.L of tumor tissue extract or 
PSA standards and add 50 .mu.L of assay buffer per well. Incubate for 3 h 
at room temperature with continuous mechanical shaking and wash x 6. Add 
100 .mu.L per well of the biotinylated polyclonal rabbit detection 
antibody diluted 1,000-fold in the polyclonal detection antibody diluent 
(100 ng of antibody per well). Incubate for 1 h as above and wash x 6. Add 
100 .mu.L per well of SA-ALP conjugate diluted 30,000-fold in the SA-ALP 
diluent (3 ng of conjugate per well). Incubate for 15 min as above and 
wash x 6. Add 200 .mu.L/well of the DFP substrate diluted 10-fold just 
before use in the substrate buffer (working DFP substrate solution is 1 
mmol/L) and incubate for 10 min at room temperature with shaking. Add 100 
.mu.L/well of the developing solution, mix by shaking for 1 min and read 
the Tb.sup.3+ specific fluorescence with the CyberFluor 614 
Immunoanalyzer. Data reduction is automatic. 
Assay of the PSA-.alpha..sub.1 -Antichymotrypsin Complex (PSA-ACT) 
This assay is exactly the same as the PSA assay described above but instead 
of using the biotinylated polyclonal rabbit anti-PSA antibody, we used the 
polyclonal rabbit .alpha..sub.1 -antichymotrypsin antibody, diluted 
500-fold in the SA-ALP conjugate diluent. We then added 100 .mu.L of a 
5,000-fold diluted FARlg-ALP conjugate (20 ng per well) and incubated for 
30 min with shaking. After washing x 6, we completed the assay by adding 
the DFP substrate as described in the PSA assay. No effort was made to 
calibrate this assay because of the unavailability of standard PSA-ACT 
complex. 
PSA was also measured in selected tumor extracts with commercially 
available kits (a). The Hybritech Tandem.RTM.-R PSA kit (Hybritech Inc, 
San Diego, Calif. 92126), (b). The IRMA-Count.RTM. PSA kit (Diagnostic 
Products Corp., Los Angeles, Calif. 90045) and (c). The Abbott IM.sub.x 
.RTM. automated PSA method (Abbott Laboratories, Chicago, Ill., U.S.A.). 
High performance liquid chromatography was performed with a Shimadzu 
system with an absorbance monitor at 280 nm (Shimadzu Corp., Kyoto, 
Japan), isocratically, using a mobile phase of 0.1 mol/L NaH.sub.2 
SO.sub.4 --0.1 mol/L NaH.sub.2 PO.sub.4, pH 6.80. Flow rate was 0.5 
mL/min. The gel filtration column used was a Bio-Sil SEC-400, 600 
mm.times.7.5 mm (BioRad Labs, Richmond, Calif.). The column was calibrated 
with a molecular weight standard solution from BioRad, containing 
thyroglobulin (670 KD), IgG (158 KD) ovalbumin (44 KD), myoglobin (17 KD) 
and cyanocobalamin (1.4 KD). Fractions of 0.5 mL each were collected with 
a fraction collector, Model FRAC-100 (Pharmacia, Uppsala, Sweden) after 
injecting a 150 mL sample. 
Statistical Analysis 
The chi-square (X.sub.2) test was used to determine the statistical 
significance of differences in distributions and all chi-square values and 
the corresponding P values were calculated by the statistical software SAS 
(SAS Institute Inc., Cary, N.C., USA). 
p53 Measurement 
Solutions and Reagents 
Lysis buffer: 150 mM CaCl, 20 mM Tris, 1% Nonidet P-40. 0.5 mM 
phenylmethysulfonylchloride (PMSF). 1 .mu.g ml.sup.-1 leupeptin. 50 g 
ml.sup.-1 aprotinin. Sample diluent (diluent for cell lysates, serum, 
polyclonal anti-p.sup.53 rabbit antiserum and alkaline 
phosphatase-conjugated goat anti-rabbit antibody): 50 mM Tris, pH 7.40, 
containing 60 g bovine serum albumin (BSA) and 1 g sodium azide per liter. 
Monoclonal anti-p.sup.53 antibody diluent; 50 mM Tris, pH 7,40, containing 
60 g bovine serum albumin, 1 g sodium azide and 0.5 mol KCl per liter. 
Substrate buffer 0.1M Tris, pH 9.1, 0.15M NaCl, 1 mM MgCl. Developing 
solution: 2.times.10.sup.-5, TbCl.sub.3, 3.times.10.sup.-3 EDTA. 0.4M 
NaOH, 1M Tris base (no Ph adjustments). Prepare as described elsewhere 
(32). Wash solution: Distilled water. Coating buffer: 50 mM Tris, pH 7.80, 
containing 1 g of sodium azide per liter. The phosphate ester of 
5-fluorosalicylic acid (FSAP) was obtained from CyberFluor Inc., Toronto, 
Canada. It is stored as a 10 mM stock solution in 0.1 M NaOH at 4.degree. 
C. for many months. This stock is diluted 10-fold in the substrate buffer 
just before use. All other chemicals were from Sigma Chemical Co., St. 
Louis, Mo., USA, except Nonidet P-40 (Boehringer-Mannheim, Indianapolis, 
Ind., USA) TbCl.sub.3.6H.sub.2 O IGFS Chemicals, Columbus, Ohio, USA) and 
the biotinylation reagent NHS-LC-Biotin (Pierce Chemical Co., Rockford, 
Ill., USA). 
p53 Standards 
Recombinant mutant human p53 protein standards in the range from 0.25-4 ng 
ml.sup.-1 were obtained from Oncogene Science, Inc., Uniondale, N.Y., USA 
and were considered the primary standards. These standards were used to 
optimize the assay and standardize cell lysates for subsequent studies. 
Another human wild-type recombinant p53 solution, prepared as described 
elsewhere (33) was a gift to us by Dr. C. Prives, Columbia University. 
This p53 preparation was diluted in the sample diluent to make standard 
solutions. 
Antibodies 
The mouse anti-p53 monoclonal antibodies, PAb 421 and PAB 240 were kindly 
provided by Dr. S. Behchimol, Ontario Cancer Institute. These are tissue 
culture supernatants containing approximately 30 .mu.g ml.sup.-1 antibody. 
The rabbit polyclonal anti-p53 antibody, CM-1, was obtained from Dimension 
Labs, Mississauga, Ontario, Canada. The goat anti-rabbit antibody, 
conjugated to alkaline phosphatase and the goat anti-mouse antibody, 
F.sub.c specific, both approximately 1 mg ml.sup.-1, were obtained from 
Jackson Immunoresearch, West Grove, Pa., USA. 
Immunoassay of p53 
White, opaque, 12-well microtiter strips (Dynatech Labs, Alexandria, 
Calif.&lt;USA) were coated with a goat anti-mouse antibody by pipetting 100 
.mu.l 500 ng well.sup.-1 of the antibody solution in the coating buffer. 
After overnight incubation at room temperature, the wells were washed four 
times with distilled water. The wells were then blocked by pipetting 200 
.mu.l well of the sample diluent, incubating for 1 h and washing as above. 
The wells were then used for the assay as follows. We add 100 ng 
well.sup.-1 of mouse monoclonal anti-pt3 antibody (PAb 421 or PAb 240) and 
50 .mu.l of sample (p53 standards of cell lysates). The antibodies are 
cell culture supernatants containing about 30 .mu.g ml.sup.-1 of antibody 
and they were diluted x 20 in the monoclonal anti-p53 antibody diluent. 
The cell lysates were used in different dilutions in the sample diluent, 
varying from 10-1000-fold. After 3 h incubation with shaking at 37.degree. 
C., the plates were washed x 4. We then added 100 .mu.l well.sup.-1 of the 
polyclonal rabbit anti-p53 antibody (diluted 5000-fold in the sample 
diluent) and incubated with shaking for 1 h at room temperature. After 
washing x 4, we added 100 .mu.l well.sup.-1 of the goat anti-rabbit 
alkaline phosphatase conjugate solution (diluted 5000-fold in the sample 
diluent) and incubated with shaking for 1 h at room temperature. The 
strips were washed again x 4 and 100 .mu.l well.sup.-1 of the FSAP 
solution (10.sup.-3 M in the substrate buffer were added and incubated for 
10 min with shaking at room temperature. The fluorescent complex was then 
formed by adding 100 .mu.l well.sup.-1 of the developing solution followed 
by brief mixing for 1 min. Time-resolved fluorometric measurements at 615 
nm were performed on the CyberFluor 615 Immunoanalyzer. Data reduction and 
plotting of calibration curves was automatic through the analyzer 
software. 
Detection of PSA mRNA 
Detection of PSA mRNA can be accomplished by the method of Deguchi et al 
(34) or a modification of it. This method involves isolation of total RNA 
or mRNA from tumors, synthesis of cDNA by reverse transcription and PCR 
amplification of the cDNA using PSA specific primers. The sequence of 
primers used are as follows: 
EQU 5'-TCG-GCA-AGT-TCA-CCC-TCA-3' 
EQU 5'-CCC-TCT-CCT-TAC-TTC-ATC-C-3'. 
PCR amplification produces a fragment of 754 base pairs which is 
electrophoresed on agarose gels and Southern blotted to Hybond N+ 
membrane. A probe (5'-GGA-ACC-TTG-GAA-ATG-ACC-AG-3') labeled with 
fluorescein is added to hybridize with cDNA for PSA. The probe is detected 
using chemiluminescence reagents from Amersham International. 
Breast Cancer Survival and ER-negative, PSA-positive Study 
One hundred and seventy four patients with primary breast cancer were 
included in this study. All patients were treated and followed at the 
Department of Gynecologic Oncology at the University of Turin. Ages of 
these patients ranged from 25 to 91 years with a median of 56 years. 
Thirty two percent of the patients were &lt;50 years and 69% &gt;50 years. The 
follow-up time ranged from 7 to 67 months with a median of 33 months. 
Clinical and pathological information, including clinical stage, 
histological cell type and grade, axillary node involvement, tumor size, 
presence of ER and PR in tumor cells and adjuvant treatment after surgery, 
was collected for each patient. According to the TNM staging system, 45%, 
47% and 8% of the patients had stage I, II and III or IV, respectively. 
Each breast cancer specimen was also histologically graded and typed. 
Thirty nine percent of patients had low grade (I), 42% had moderate grade 
(II), and 19% had high grade (III). Seventy percent of patients had ductal 
carcinomas. The rest had lobular (13%), lobular in situ (2%), medullary 
(5%), papillary (2%), tubular (2%), tubulo-lobular (3%), or unknown types 
(3%). In the data analysis, histological type was grouped into two 
categories, i.e. ductal versus non-ductal, because of the small number of 
patients who had types other than ductal carcinomas. 
The size of tumor in these patients ranged from 0.7 to 6 cm, and median and 
mean sizes were identical, 2.4 cm. Fiftyone percent of the patients had 
tumor invading the axillary lymph nodes. Of the 174 patients, 56% were 
treated with adjuvant therapy as follows: tamoxifen (37%), chemotherapy 
(15%), or both (4%). The rest (44%) received no further treatment after 
surgery. 
Demographic, clinical and pathological variables, including age, clinical 
stage, histological grade and type, nodal status, tumor size, ER and PR, 
and adjuvant treatment, were compared between PSA-positive and 
PSA-negative groups, using the contingency table and Chi-square test in 
order to examine the associations between PSA and these variables. The 
relationship between each of the study variables and relapse-free or 
overall survival was expressed by the hazard ratio and its 95% confidence 
interval, which was calculated univariately using the Cox proportional 
hazard regression model (35). The multivariate Cox regression model was 
also employed to evaluate the impact of PSA immunoreactivity on patient 
survival while controlling for other clinical and pathological variables 
which may also affect the survival, such as clinical stage (I, II or 
IIl/IV), nodal status (positive or negative), tumor size (greater or less 
than mean size), steroid hormone receptors (presence or absence), and 
adjuvant treatment (none, tamoxifen, or both tamoxifen and chemotherapy). 
Kaplan-Meier relapse-free and overall survival curves (36) were 
constructed to demonstrate the survival difference between PSA-positive 
and negative groups. The logrank test (37) was used to examined the 
significance of the differences between survival curves. 
Serum Samples for PSA Subfraction Study 
Three presurgical sera with total PSA values .gtoreq.50 ng/L were selected 
from a series of 198 presurgical sera of patients with primary breast 
cancer. No other criterion was used to select these three sera. A total of 
seven post-surgical sera with total PSA .gtoreq.16 ng/L were selected from 
another series of 346 breast cancer patients who were treated by surgery. 
Three normal (from non-breast cancer subjects) sera with total PSA 
.gtoreq.35 ng/L were also selected from a total of 212 sera from female 
blood donors. These were provided by the Red Cross Blood Transfusion 
Service in Toronto. Other clinical samples included sera from three normal 
male blood donors and sera from three males who underwent radical 
prostatectomy for prostate cancer. All six male sera had PSA .gtoreq.80 
ng/L. All samples were stored at -20.degree. C. 
We selected sera with total PSA .gtoreq.16 ng/L in order to be able to 
determine the PSA molecular forms by HPLC followed by PSA 
immunofluorometry. Samples with total PSA&lt;16 ng/L are not suitable because 
the individual HPLC fractions contain very little PSA which is difficult 
to measure. 
High-performance liquid chromatography (HPLC) of the Serum PSA Subfraction 
Samples 
HPLC analysis was performed with a Hewlett Packard 1050 system. The mobile 
phase was a 0.1 mol/L sodium sulphate and 0.1 mol/L sodium dihydrogen 
phosphate, pH 6.80. The gel filtration column used was a TSK-GEL G3000SW, 
60 cm.times.7.5 mm (TosoHaas, Montgomeryville, Pa. 18936) and was 
calibrated with a molecular mass standard solution from Bio-Rad (Bio-Rad 
Laboratories, Hercules, Calif. 94547). The flow rate was 0.5 mL/min and 
the HPLC was run isocratically. After injection of 100-500 uL of each 
certrifuged sample, fractions of 0.5 mL were collected and analyzed for 
PSA using the outlined method below. Sample carry over of &lt;5% was ensured 
by in between-sample-injection column and injector washings, and by the 
order of sample injection (e.g. the samples with the highest total PSA 
were injected last). 
PSA Immunoassay of the HPLC Serum Subfraction Samples 
PSA determinations were performed using a modified methodology from our 
highly sensitive and specific immunofluorometric procedure previously 
established and described in detail elsewhere (18). Briefly, the PSA assay 
uses a mouse monoclonal anti-PSA capture antibody coated to polystyrene 
microtiter wells, a biotinylated monoclonal anti-PSA detection antibody, 
and alkaline phosphatase-labeled streptavidin (SA-ALP). In this 
immunoassay, 100 uL of sample is incubated with the coating antibody in 
the presence of 50 uL of assay buffer containing the monoclonal anti-PSA 
detection antibody. After 1 h incubation followed by washing x 6, the 
SA-ALP conjugate is added for 15 min., followed by another washing x 6. 
The activity of ALP is then measured by adding the substrate 
5-fluorosalicylphosphate, incubating for 0 min. and then by adding a 
Tb.sup.3+ and EDTA-containing developing solution. After 1 min. the 
fluorescence is measured in the time-resolved fluorometric mode with the 
Cyberfluor-615 Immunoanalyzer (Cyberfluor Inc., Toronto, Ontario). This 
assay has a biological detection limit of 1 ng/L of PSA. Details are 
described elsewhere (18). All assays were run in duplicate. 
Although preferred embodiments of the invention are described herein in 
detail, it will be understood by those skilled in the art that variations 
may be made thereto without departing from the spirit of the invention or 
the scope of the appended claims. 
TABLE I 
______________________________________ 
Analysis of PSA in Breast Tumor Extracts 
PSA, mg/L 
______________________________________ 
Number of Patients 
&lt;0.05 .gtoreq.0.05 
.gtoreq.0.10 
.gtoreq.0.30 
525 374 151 96 49 
% of Samples 71.2% 28.8% 18.3% 9.3% 
______________________________________ 
TABLE II 
______________________________________ 
Relationship Between Estrogen and Progesterone 
Receptors, PSA and p53 Levels in Breast Tumor Extracts.sup.1 
______________________________________ 
Samples (N = 525) 
PSA (+) (%) 
PSA (-) (%) 
P Value 
______________________________________ 
ER (+) 393 127(32.3) 266(67.7) 
ER (-) 132 24(18.2) 108(81.8) 0.002 
PR (+) 321 111(34.6) 210(65.4) 
PR (-) 204 40(19.6) 164(80.4) &lt;0.001 
ER (+) or PR (+) 407 
131(32.2) 276(67.8) 
ER(-) and PR (-) 118 
20(16.9) 98(83.1) 0.001 
ER (+) and PR (+) 307 
107(34.8) 200(65.2) 
ER (+) and PR (-) 86 
20(23.3) 66(76.7) 
ER (-) and PR (+) 14 
4(28.6) 10(71.4) 
ER (-) and PR (-) 118 
20(16.9) 98(83.1) 0.002 
______________________________________ 
Samples (N = 558) 
p53 (+) (%) 
p53 (-) (%) 
P Value 
______________________________________ 
ER (+) 416 64(15.4) 352(84.6) &lt;0.001 
ER (-) 142 50(35.2) 92(64.8) 
PR (+) 338 47(13.9) 291(86.1) &lt;0.001 
PR (-) 220 67(30.4) 153(69.6) 
ER (+) or PR (+) 428 
68(15.9) 360(84.1) &lt;0.001 
ER (-) and PR (-) 130 
46(35.4) 84(64.6) 
______________________________________ 
Samples (N = 474) 
p53 (+) (%) 
p53 (-) (%) 
P Value 
______________________________________ 
PSA (+) 90 20(22.2) 70(77.8) P = 0.37 
PSA (-) 384 103(26.8) 281(73.2) 
______________________________________ 
.sup.1 For negativity cutoff levels see text. Values in brackets are 
percentages. 
TABLE III 
______________________________________ 
Distribution of PSA-Positive, Estrogen Receptor-Positive 
and Progesterone Receptor-Positive Tumors in Various Age Groups 
% of Positive Tumors.sup.1 
Patient Age (Years) 
PSA (+) ER (+) PR (+) 
______________________________________ 
&lt;35 N = 18) 
33.3(6/18) 50.0(9/18) 44.4(8/18) 
35-44 (N = 66) 
36.4(24/66) 
71.2(47/66) 
62.1(41/66) 
45-54 (N = 104) 
38.5(40/104) 
64.4(67/104) 
58.7(61/104) 
&gt;55 (N = 336) 
23.8(80/336) 
80.1(269/336) 
62.5(210/336) 
P Value.sup.2 
0.012 0.001 0.45 
______________________________________ 
.sup.1 In brackets are numbers of positive tumors per total number of 
tumors in each group. N = number of patients per group. 
.sup.2 P value for comparing the distribution of positive or negative 
tumors for each parameter, in the various age groups. 
TABLE IV 
______________________________________ 
Association of PSA-Positive 
Tumors with Disease Stage 
Disease Stage % of PSA-Positive Tumors.sup.1 
______________________________________ 
0 42.9(6/14) 
1 30.7(35/114) 
2 22.0(13/59) 
3 12.5(2/16) 
P 0.18 
0-1 32.0(41/128) 
2-3 20.0(15/75) 
P 0.06 
______________________________________ 
.sup.1 In brackets are numbers of positive tumors per total number of 
tumors in each group. 
TABLE V 
______________________________________ 
Relationship between PSA Immunoreactivity and ER & PR 
Receptor 
No. of No. of 
Status Patients PSA + OR & 95% CI 
p value 
______________________________________ 
ER-;PR- 226 32(14%) 1.00 
ER+,PR- 139 28(20%) 1.53(0.88-2.67) 
0.13 
ER-,PR+ 58 24(41%) 4.28(2.25-8.14) 
&lt;0.01 
ER+,PR+ 852 302(35%) 3.33(2.23-4.96) 
&lt;0.01 
______________________________________ 
OR: Odds ratio. 
CI: Confidence interval. 
TABLE VI 
__________________________________________________________________________ 
Associations between PSA and relapse-free survival 
stratified by the status of estrogen receptors 
95% 
ER PSA(+) 
Hazards 
confidence 
status patients ratio 
Interval 
value P 
__________________________________________________________________________ 
ER cutoff at 10 fmol/mg 
Univariate analysis 
ER(+) (n = 112).sup.2 
36 0.98 0.37-2.61 0.97 
ER(-) (n = 57) 
9 0.16 0.02-1.22 
0.08 
Multivariate analysis.sup.3 
ER(+) (n = 112) 
36 0.80 0.27-2.32 0.68 
ER(-) (n = 57) 
9 0.13 0.02-1.15 
0.07 
ER cutoff at 20 fmol/mg 
Univariate analysis 
ER(+) (n = 95) 
27 1.42 0.46-4.34 0.54 
ER(-) (n = 74) 
18 0.18 0.04-0.76 
0.02 
Multivariate analysis 
ER(#) (n = 95) 
27 0.96 0.27-3.33 0.94 
ER(-) (n = 74) 
18 0.20 0.04-0.93 
0.04 
__________________________________________________________________________ 
.sup.1 The ratio of hazards between PSApositive and PSAnegative patients. 
.sup.2 N = number of patients. 
.sup.3 Adjusted for age, clinical stage, nodal status tumor size, and 
histological grade. 
TABLE VII 
__________________________________________________________________________ 
Clinical samples used in this study 
PSA LEVEL 
TIME 
CASE ID.sup.(1) 
SERUM TYPE GENDER 
in ng/L 
OF 
__________________________________________________________________________ 
A normal; non-breast cancer 
female 
36 Ra 
B normal; non-breast cancer 
female 
50 Ra 
C normal; non-breast cancer 
female 
80 Ra 
D breast cancer; pre-surgical 
female 
54 &lt;1 month 
E breast cancer; pre-surgical 
female 
59 &lt;1 month 
F breast cancer; pre-surgical 
female 
82 &lt;1 month 
G.sup.(2) 
breast cancer; post-surgical 
female 
61 163 
month 
H breast cancer; post-surgical 
female 
65 92 months 
I breast cancer; post-surgical 
female 
63 36 months 
J breast cancer; post-surgical 
female 
53 104 
month 
K breast cancer; post-surgical 
female 
50 1 month 
L breast cancer; post-surgical 
female 
16 36 months 
M breast cancer; post-surgical 
female 
101 1 month 
N normal; non-prostate cancer 
male 413 Ra 
O normal; non-prostate cancer 
male 554 Ra 
P normal; non-prostate cancer 
male 544 Ra 
Q.sup.(3) 
post-radical prostatectomized 
male 84 7 months 
R post-radical prostatectomized 
male 132 10 months 
S post-radical prostatectomized 
male 420 21 months 
__________________________________________________________________________ 
i. The volume injected into the HPLC column was .about.500 uL for all 
samples with the exception of cases D, E, F, and J which were 200 uL, 100 
uL, 100 uL, and 460 uL, respectively. 
ii. Patients I to M are still in remission. For patients G and H no 
current clinical status was available. 
iii. Patients Q, R, S are still clinically asymptomatic but biochemically 
relapsed. 
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