Assay for PSP94 protein

PSP94 occurs in the serum mainly as a complex with carrier protein. Determination of bound PSP94 provides an indicator of prognosis in prostate cancer and assists in diagnosis of prostate cancer in patients with borderline elevations of PSA.

FIELD OF THE INVENTION 
This invention relates to methods for determining proteins which are 
markers for prostatic disease, including prostate tumours. 
BACKGROUND OF THE INVENTION 
Prostate cancer is one of the most common cancers and its incidence is 
increasing. It is desirable to be able to diagnose prostate cancer at an 
early stage when the disease is potentially curable. A number of prostate 
diseases, including prostate cancer, lead to increased blood levels of 
prostate-derived proteins such as prostate specific antigen (PSA) and 
these proteins serve as diagnostic markers for these diseases. Blood 
levels of PSA, for example, are increased when normal prostatic structure 
is disrupted by benign or malignant tumours or by inflammation. 
Testing of PSA has increased rapidly among asymptomatic men in North 
America, and recommendations favouring such screening have been issued by 
the American Cancer Society and the American Urological Association. About 
70% of patients with cancers identified by digital rectal examination, 
transrectal ultrasound, and needle biopsy have PSA concentrations above 
the cut-off values generally used. About 70-80% of abnormal PSA values in 
asymptomatic men, however, are false positives; only 1 in 4 men with a PSA 
above 4 .mu.g/L will have prostate cancer (Catalona et al., (1991), New 
England Journal of Medicine, 324, 1156-1161; Chadwick et al., (1991), 
Lancet, 338, 613-616). The diagnostic potential of PSA measurements in 
serum is limited by the increase of PSA concentrations in many subjects 
with benign prostatic hyperplasia (BPH). 
PSA has been shown to exist in serum in complexes with antichymotrypsin 
(ACT), .alpha..sub.2 macroglobulin (AMG), protein C inhibitor and 
pregnancy zone protein. 
When the ratio of PSA-ACT complex: total PSA was determined, 38-60% of 
false positives in the range 2.5-25 .mu.g/L were eliminated, giving 
improved diagnostic accuracy without loss of sensitivity. 
Measurement of serum PSA is not, however, totally satisfactory for 
diagnosis of prostate disease and the use of multiple markers has been 
suggested. 
PSA (also known as .alpha.-microseminoprotein) and PSP94 (prostate 
secretory protein of 94 amino acids, also known as 
.beta.-microseminoprotein) are the two most abundant secretory proteins 
from prostate and PSP94 has been examined by a number of groups as a 
possible blood marker for prostate disease, using radioimmunoassay, enzyme 
immunoassay, two site immunoradiometric assay and enzyme linked in 
immunosorbent assay. 
These studies have produced conflicting results on the efficacy of serum 
PSP94 as a marker for prostate cancer (van der Kammer et al., (1993), 
Urol. Res., 21, 227-233). 
SUMMARY OF THE INVENTION 
The inventors have shown that most of the PSP94 in the blood occurs in a 
tightly protein-bound form. It is believed that previous assays have 
measured only free serum PSP94, which has been found by the inventors not 
to correlate significantly with medical outcome after treatment in 
prostate cancer. 
This invention enables methods to determine the level of bound PSP94 or 
total PSP94 in biological fluids such as serum, plasma and seminal fluid. 
The inventors have shown that the level of bound PSP94 in serum is a 
better predictor of the likelihood of relapse-free survival after 
radiation treatment in prostate cancer than the level of free PSP94, a 
higher level of bound PSP94 being associated with a poorer prognosis.

DESCRIPTION OF THE INVENTION 
The present inventors have found that PSP94 protein exists in human blood 
both as free PSP94 protein and as a complex of PSP94 protein bound to 
carrier protein. 
The bound form of PSP94 is surprisingly stable, similar to bound forms of 
PSA, and is very resistant to dissociation. 
Furthermore, most of the PSP94 protein present in human blood is in the 
protein-bound form, with only a small percentage of total serum PSP94 in 
free form. 
It has been shown by the inventors that none of 15 monoclonal antibodies 
and one polyclonal antibody raised using purified PSP94 as antigen could 
recognise bound PSP94 (data not shown). It is believed that the PSP94 
epitopes recognised by these antibodies are inaccessible in the bound 
PSP94 complex. 
Previously described assays for PSP94, therefore, measure only free serum 
PSP94 protein, giving a false picture of the complete status of PSP94 in 
blood. 
The inventors have found that free serum PSP94 levels do not serve as good 
indicators of medical outcome in prostate cancer, and a truer picture is 
obtained by determining serum protein-bound PSP94 (correlation between 
relapse-free survival and free PSP94, on univariate analysis: p=0.8; for 
bound PSP94:p=0.075). 
As used herein, determination or measurement of "bound PSP94" in a 
biological fluid refers to determination of the portion of PSP94 which 
occurs in the fluid bound to a carrier protein. "Bound PSP94" may be 
determined while still bound to carrier protein, for example, by an 
immunoassay using a monoclonal antibody specific for bound PSP94, as 
described more fully below, or by dissociation and determination of the 
released free PSP94. 
Table II shows that higher pretreatment levels of serum bound PSP94 were 
associated with an increased rate of relapse following radiation treatment 
in patients receiving radical radiotherapy for non-metastatic (T1-T4NXMO) 
prostate carcinoma (multivariate analysis: p=0.022). 
The present invention provides methods for determining bound PSP94 in 
biological fluids such as serum, plasma or seminal fluid. Measurement of 
serum bound PSP94 may be used as a screening test for the detection of 
prostate cancer or may be used, in established cases of prostate cancer, 
as a predictor of outcome or of likelihood of successful treatment. 
The invention also provides a method of improving the accuracy of diagnosis 
of prostate cancer in patients presenting with a borderline elevation (&lt;10 
ng/ml) of serum PSA. It has also been shown that in patients with 
borderline PSA elevation, serum bound PSP94 is a good predictor of 
relapse-free survival after treatment, whereas PSA values did not 
correlate with relapse-free survival. 
It is contemplated that measurement of serum bound PSP94 may be used in 
addition to PSA level screening, to investigate further patients with 
borderline PSA elevation, a large percentage of whom are known to be false 
negatives with respect to prostate cancer. 
In one embodiment, bound PSP94 in a biological fluid, for example serum, is 
determined by first separating from the fluid the PSP94 which is complexed 
with carrier protein, followed by dissociation of the complex and 
determination of the released free PSP94. The complex is separated, for 
example, by a molecular sieve technique. The complex is then dissociated 
and free PSP94 separated from other components by SDS-polyacrylamide gel 
electrophoresis in the presence of .beta.-mercaptoethanol. PSP94 is 
identified on the gel using a labelled anti-PSP94 antibody and the signal 
from the antibody label is quantified by comparison with known amounts of 
purified PSP94 run in parallel. 
In a further embodiment, bound PSP94 in a biological fluid is determined by 
first dissociating any PSP94/carrier protein complex with a suitable 
dissociating agent, followed by separation and determination of the 
released free PSP94. For example, the released PSP94 may be separated by 
centrifuging the dissociation mixture through a molecular 
sieve/PSP94-affinity matrix column to trap the PSP94 in the matrix. PSP94 
is then eluted from the matrix and determined, for example by immunoassay. 
In a further embodiment, bound PSP94 is determined without dissociation 
from its complex with carrier protein, for example by immunoassay using an 
antibody which binds selectively to protein-bound PSP94 and not to free 
PSP94. Similar methods have been described for immunoassay of 
protein-bound PSA (for example U.S. Pat. No. 5,501,983 and Chen et al., 
(1995), Clin. Chem., 41, 1273-1282, incorporated herein by reference) and 
one of ordinary skill in the art can similarly determine bound PSP94. 
The invention further provides a means of determining total serum PSP94 or 
of the relative levels of free and bound PSP94 in serum. 
It has also been found by the inventors that in a few patients examined 
after the initiation of hormone therapy, serum bound PSP94 remained in the 
range found in pre-treatment prostate cancer patients. This contrasts with 
serum PSA, the levels of which become negligible once hormone therapy is 
initiated. PSP94 may provide an androgen independent prostate cancer 
marker useful for monitoring the success or failure of hormone therapy in 
prostate cancer patients. 
EXAMPLES 
The examples are described for the purposes of illustration and are not 
intended to limit the scope of the invention. 
Methods of protein and peptide biochemistry and immunology referred to but 
not explicitly described in this disclosure and examples are reported in 
the scientific literature and are well known to those skilled in the art. 
Example 1 
Free and Bound PSP94 Protein in Serum 
Materials and Methods 
Patient serum. Blood (.about.10 ml) was collected after induction of 
anaesthesia from patients undergoing radical prostatectomy for prostate 
cancer, radical cystoprostatectomy for bladder cancer and radical 
nephrectomy for kidney cancer. Blood samples were taken without EDTA or 
heparin treatment. After clotting, samples were centrifuged briefly to 
separate serum. For large scale purification, larger amounts of blood were 
collected during surgery but prior to any transfusion. Because these 
latter samples may contains small amounts of body fluids, 10 ml blood 
samples were taken before surgery and used as a control. 
Purification and biotinylation of human PSP94 from semen. Human PSP94 was 
purified from semen samples according to the protocol reported previously 
(24) with the following modifications; only one round of ammonium sulfate 
precipitation from 30 to 70% saturation was conducted, and the use of HPLC 
anion exchange column was eliminated. The purification of PSP94 was 
characterized by overloading (.about.20 .mu.g) on a 15% SDS-PAGE without 
any other visible bands after Coomassie blue R-250 staining. For 
biotinylation and detection of the labelled free form of PSP94, a 
Biotin-Blot Protein Detection Kit (BioRad, Mississauga, Ontario Canada) 
was used. The biotinylation reaction was initiated by adding 9.75 .mu.l of 
NHS-Biotin (N-hydroxysuccinide biotinate in dimethylformamide, 75mM) to 1 
mg PSP94 in 0.1 M sodium borate buffer pH 8.8 in a total volume of 350 
.mu.l, followed by incubation at room temperature for 4 hours. 20 .mu.l of 
1 M NH.sub.4 Cl was added and incubated at room temperature for 10 min. 
Biotin-labelled PSP94 was desalted by 3 rounds of spinning/dilution using 
a Centricon-3 cartridge (Amicon, Beverly, Mass.). For detection of 
biotin-PSP94, ECL-Western blotting (ECL Western Blotting Kit, Amersham, 
Oakville, Ontario Canada) was used. The transferred blot (ECL-Hybond 
nitrocellulose membrane, from Amersham) with biotin-PSP94 was first 
reacted with avidin-HRP (horseradish peroxidase) conjugate at 1:15,000 
dilution in 0.5% blocking regent (Boehringer Mannheim, Laval, Quebec 
Canada)/TBS (50 mM Tris-HCl pH7.5, 150 mM NaCl). ECL detection was 
performed according to the manufacturer's instruction (Amersham). The 
efficiency of biotinylation labelling of PSP94 was titrated by a dot blot 
test using the same procedure. Labelling efficiency was such that as low 
as 10 ng of the labelling mixture bound to ECL-Hybond membrane was 
detectable. However, using the HRP colour development reagent (BioRad), 
sensitivity of detection was reduced at least ten fold. 
SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The Laemmli system was 
used (25). 15% PAGE (30%T 2.67%C) was prepared according to the BioRad 
protocol. For samples of high density serum proteins, a high concentration 
of SDS (1%) and reducing agent (2.5% .beta.-mercaptoethanol) was used to 
dissociate serum proteins. For the purified serum proteins, standard 
sample dye(25) at a final concentration of SDS (0.4%) glycerol (10%) and 
Tris-HCl (0.3 M pH 6.8), and reducing agent (1% .beta.-mercaptoethanol) 
was used. 
Native PAGE. The dicontinuous buffer system of Ornstein-Davis 
(Tris/chloride/glycine) was used (26). 15% polyacrylamide gel (30%T 2.67%C 
) was prepared using a BioRad mini-protein II system. A pH 8.8 electrode 
buffer was used. Serum samples were mixed with non-denaturing, bromophenol 
blue sample dye at a final concentration of 10% glycerol, 60 mM Tris 
pH6.8, and loaded directly. 
Molecular sieve column purification. 1.5 ml of serum was applied to 30 ml 
Sephacryl S-200HR (Pharmacia, Montreal, Quebec Canada) packed in a 
45.times.1.5 cm column (Bio-Rad Econo-column), and eluted by PBS 
(phosphate buffered saline). Fractions of 0.5 ml were collected at a flow 
rate of 0.1 ml/min. All fractions were monitored by OD280.sub.nm. About 10 
.mu.l of sample was taken from each fraction tube for Western blotting 
analysis. 
Protein A affinity column purification. The low salt method of protein A 
affinity chromatography was followed (27). Fractions of Peak I after 
molecular sieve separation of serum total protein were pooled, pH was 
added to .about.8.0 by adding 1/10 volume of 1.0 M Tris (pH 8.0) and 
applied to a protein A column (Gibco/BRL, Burlington, Ont). The 
pass-through portion (with no affinity for the protein A matrix) was 
collected, immediately after the void volume was passed. About 5 column 
volumes of PBS were used for washing, and the washing solution was saved. 
The bound IgG portion was eluted from the column by 100 mM glycine (pH 3.0 
) in a stepwise fashion with 500 .mu.l per fraction of 100 mM glycine (pH 
3.0) and neutralized immediately in 50 .mu.l of 1 M Tris (pH 8.0). All IgG 
containing fractions were monitored by OD280.sub.nm and pooled. 
Western blotting. The chemiluminescence procedure was performed using an 
ECL Western Blotting Kit (Amersham) according to the protocol provided by 
the manufacturer. The primary antibodies were from rabbit antiserum to 
human PSP94 purified from seminal plasma, and were gifts from two sources, 
Dr. Michel Chretien (19) and Dr. S. Garde. Secondary antibodies, HRP 
(horse radish peroxidase) conjugated antiserum against rabbit IgG, were 
purchased from either Amersham or Dimension Laboratories, (Mississauga, 
Ontario, Canada). The first and second antibodies were diluted 5,000 and 
1,000 times respectively, as described previously (21). Stripping of 
previous Western blotting signals was in a buffer of 50 mM 
.beta.-mercaptoethanol, 2% SDS, 62.5 mM Tris -HCl pH 6.7, and incubated at 
room temperature for 1 hour. The stripping was tested after reaction with 
ECL detection buffer. 
In vitro dissociation of serum proteins. Serum samples (5 .mu.l) were 
treated with the following reagents (final concentration) at room 
temperature for 3 minutes; SDS(1%), SDS (1% ) and .beta.-mercaptoethanol 
(2.5%), SDS (1%) and .beta.-mercaptoethanol (2.5%) plus boiling, 1.5 M 
urea 50 mM Tris pH7.5, 3 M urea 50 mM Tris pH 7.5, 1.5 M guanidine 
chloride and 3 M NaCl. For purified PSP94-bound complex preparations (60 
.mu.l), treatments were: SDS (0.4%), SDS (0.4%) and .beta.-mercaptoethanol 
(1%), SDS (0.4%) and .beta.-mercaptoethanol (1%) plus boiling, 1.5 M urea 
50 mM Tris pH7.5, 3 M urea 50 mM Tris pH 7.5, 1.5 M guanidine chloride and 
3 M NaCl. The denatured samples were analysed immediately by native PAGE 
and Western blotting experiments. 
RESULTS 
Native and SDS-PAGE analysis of total serum proteins from prostate cancer 
patients. In order to differentiate various forms (free or bound forms) of 
PSP94 in serum, two kinds of PAGE (polyacrylamide gel electophoresis) were 
employed. Native, non-SDS and non-denaturing PAGE was used to separate 
serum proteins in their original forms. SDS-PAGE was used with serum 
samples treated by reducing agent (.beta.-mercaptoethanol) plus boiling to 
completely dissociate all the bound protein in serum. Two identical sets 
of serum samples (10 .mu.l each) from patients undergoing radical 
prostatectomy and radical nephrectomy were analysed by Western blot 
experiments to identify PSP94. Control samples were from seminal plasma, 
natural (nPSP94) and recombinant GST-PSP94. Results are shown in FIG. 1. 
No PSP94 band corresponding to the size of the free form of purified PSP94 
was found in the serum samples, when samples were run on a native, non-SDS 
gel (FIG. 1A). This indicates that in these sera from patients with 
different cancers, free PSP94 is present at very low levels. It may be 
less than 1 ng/10 .mu.l, i.e. 100 ng/ml, based on comparison with the 
nPSP94 lane, where 1 ng was loaded. FIG. 1B is the parallel experiment but 
with the samples treated with SDS, .beta.-mercaptoethanol and boiling. A 
free PSP94 band was observed in all tumor serum samples, suggesting that 
most of PSP94 present in serum is in a bound form and can be dissociated 
by detergent and reducing agents. No PSP94 was detectable in urine. 
Molecular sieve separation of PSP94 bound complexes from serum. Since 
native and SDS-PAGE analyses indicated the existence of serum PSP94-bound 
complexes, molecular sieve (Sephacryl S-200HR) column chromatography was 
used to separate and purify these complexes, The elution was in PBS buffer 
and under non-denaturing condition. To identify bound PSP94, in FIGS. 2 
and 3, fractions of peak I, the proposed PSP94-bound complexes, always 
eluted with or near IgG fractions in molecular sieve column 
chromatography. In order to purify and separate serum PSP94-bound protein 
complexes from IgG, peak I fractions from molecular sieve column 
separation were pooled and further purified by a protein A column. FIG. 4 
shows the results of the analysis after one round column purification. 
FIG. 4 (top) shows Coomassie blue staining of PAGE analysis of this 
experiment. The two strong bands above PSP94 at .about.25 and 55 kDa are 
likely to be IgG light and heavy chains, which have been dissociated by 
reducing agents plus boiling treatments. After protein A column 
chromatography, most of the IgG in peak I (before column) was in the 
eluate portion, and IgG remaining on the protein A column portion was low. 
Western blotting analysis (FIG. 4 middle) of this PAGE showed that most of 
the PSP94 signal was in the pass-through column portion, as compared with 
the PSP94 bands shown before column purification. This experiment implies 
that most of the serum IgG does not bind to serum PSP94 and most of serum 
PSP94-bound complexes showed no affinity with a protein A columns. 
As shown in FIGS. 2, 3 and 4, Western blot analysis of serum proteins 
always had high background of non-specific signals. In these blots, 
multiple positive bands at .about.25, 55, 70 kDa were repeatedly observed 
at higher intensity than PSP94 bands. None of the peak areas of these 
positive bands overlapped completely with the PSP94 peak 1. From the 
intensity of these bands, and also from the position of their appearances 
in the fractionation, we suppose that they represented albumin, and the 
two chains of immunoglobulins (IgG), since these two most abundant 
components always showed overloading bands in PAGE analysis. The results 
of protein A purification (FIG. 4 top and middle) also suggested that 
these non-specific signals might be due to the cross-reaction of two 
chains of IgG with polyclonal antibodies used in Western blotting 
experiments, since most of the IgG bands remain unchanged in the elute and 
were decreased in the pass-through and wash portions from the protein A 
column. To determine whether the first or second polyclonal antibodies 
were responsible for this non-specific binding, signals of Western 
blotting experiment of FIG. 4 (middle) was completely stripped and reacted 
with only second antibody (HRP-conjugated swine against rabbit IgG). The 
result of this control experiment is shown in FIG. 4 (bottom). Since the 
two experiments have the same background signal except for the PSP94 
bands, we conclude the second antibody cross-reacts with human IgG. This 
cross-samples from fraction tubes were denatured by boiling with SDS-PAGE 
dye and tested by SDS-PAGE and Western blotting experiments as FIG. 1. The 
size of free PSP94 band is indicated by positive controls (PSP94 from 
seminal fluid). The specificity of rabbit antiserum against PSP94 in human 
seminal plasma is indicated by short time exposure of the Western blots, 
in which only PSP94 monomer and its remaining dimer are visible (shown in 
first lane, FIG. 2). FIG. 2 shows two peaks of PSP94-containing fractions 
in serum; the first peak (peak I) was found in fractions #32-38, with 
higher molecular weight, and the second peak (peak II) was located at 
lower molecular weight in fractions #52-59 with very strong PSP94 
immunoreactive activity in this PCa patient serum. Peak I is considered to 
include serum PSP94-bound complexes, since peak I proteins are larger in 
size than serum albumin (67 kDa in fractions #44-50), which is the most 
abundant component in serum (60%) and appeared as an overloaded band. The 
fractions eluted just before the region where IgG (.about.150 kDa), the 
second most abundant (10-20%) protein in serum was eluted. The first peak 
has been repeatedly observed in 6 serum samples from 7 PCa patients. Peak 
II was observed as a strong signal in only in 1 of 7 PCa samples, and this 
sample was thus selected for analysis in FIGS. 2 and 3. 
In order to determine if peak II represents free PSP94, we used 
biotinylated PSP94 as an indicator to monitored molecular sieve separation 
of total serum proteins (FIG. 3). A large amount (15 .mu.g) of biotin 
labelled PSP94 was loaded together with 1.5 ml serum onto a Sephacryl 
S-200HR column. Fractionation and Western blotting were performed as in 
FIG. 2. To differentiate free (biotinylated) PSP94 from the bound form in 
molecular sieve chromatography, two identical blots were assessed using 
either polyclonal anti-PSP94 antiserum (FIG. 3A) or HRP (horseradish 
peroxidase)-avidin and ECL (enhanced chemiluminescence) reaction (FIG. 
3B). Two peaks of PSP94 bands showed high (peak II) and lower (peak I) 
immunoreactivity to PSP94 polyclonal antiserum as in FIG. 2, however, the 
intensity of signals of peak II in FIG. 3 is higher than in FIG. 2, 
indicating that this peak contains both free forms of natural and 
biotinylated PSP94. This result is confirmed by the result of blot (FIG. 
3B) reacted with HRP-avidin and ECL analysis of Biotin-PSP94. Only one 
peak (peak I) was detected in this blot. It is thus mostly likely that 
peak I represents PSP94-bound complexes. 
Protein A affinity column purification of serum PSP94-bound complexes. As 
shown reactivity was found in two commercially available second 
antibodies: swine anti-rabbit and goat anti-rabbit, consistant with high 
levels of conservation of the IgG gene in evolution among mammals 
including human. 
Stability of serum PSP94-bound complexes. To test the nature of the binding 
of PSP94 with serum proteins, serum samples from cancer patients were 
treated by several denaturing chemical regents: 1% SDS, 1% SDS, 1% 
.beta.-mercaptoethanol, 1% SDS 1% .beta.-mercaptoethanol plus boiling, 
high concentrations of urea (1.5 to 3 M), 1.5 M guanidine hydrochloride 
(GHCL), and 3 M NaCl. FIG. 5A shows that the binding of PSP94-bound 
complexes is very stable, with chemical resistance to most of these 
denaturing treatments. Only strong reducing reagents (1% SDS, 1% 
.beta.-mercaptoethanol plus boiling) effectively dissociated the bound 
complexes. In order to confirm results obtained from whole serum samples, 
crude preparations of serum PSP94-bound complexes, purified by molecular 
and two rounds of protein A column purification (shown in FIG. 5B), were 
repeatedly tested with similar results. 
Example 2 
Assay for Serum Total PSP94 Protein 
PSP94 Affinity Matrix: 
CNBr-activated Sepharose 4B (Pharmacia) is used to immobilise an antibody 
specific for PSP94 protein in accordance with the manufacturer's protocol, 
to give a PSP94 affinity matrix. Suitable antibody can be prepared by 
conventional techniques using purified PSP94 protein, for example from 
seminal fluid, as antigen. 
Spin Column Cartridge 
A spin column cartridge is prepared with an upper layer of Sephadex G50 and 
a lower layer of PSP94 affinity matrix prepared as described above. 
Method 
Step 1: Dissociation 
A 1 ml serum sample is treated with SDS and .beta.-mercaptoethanol at 
100.degree. C. for about 3 minutes to dissociate PSP94 from its bound 
form. 
Step 2: Separation of dissociated PSP94 
The dissociated sample is applied to a spin column cartridge (as described 
above) and centrifuged briefly. 
SDS and .beta.-mercaptoethanol are trapped in the upper Sephadex layer, 
while the dissociated serum proteins pass through to the lower layer, 
where free PSP94 protein is adsorbed by the affinity matrix. 
After the first centrifugation, the upper Sephadex layer is removed and the 
lower layer is washed extensively with phosphate-saline buffer. 
The affinity matrix layer is then resuspended in a suitable buffer to elute 
PSP94 protein from the matrix, which is removed by a further 
centrifugation. 
The concentration of PSP94 protein is then determined in the eluate by a 
conventional protein determination method, for example, immunoassay or 
OD.sub.280. 
Example 3 
Assay of Free and Bound Serum PSP94 
In order to determine the ratio of free/bound or free/total PSP94 in serum, 
total PSP94 can be determined as described in Example 2, free PSP94 can be 
determined by conventional methods, as previously described, for example, 
in Xuan et al., (1996) J. Cell Biochem., 63, 61-73) (which is incorporated 
herein by reference) and bound PSP94 can be determined by subtraction. 
Example 4 
Materials and Methods Patient Selection 
From a group of patients receiving radiotherapy for prostate cancer at the 
London Regional Cancer Clinic between 1991-1992, 44 met the following 
criteria: T1-T4NXMO prostate cancer, treated with radiotherapy alone, 
pretreatment PSA available and archived pretreatment sera available for 
PSP94 determinations. 
Patient Treatment 
All patients were treated with curative intent with radiotherapy. A 
standard four field box technique, using high energy (18-25 Mv) photons 
encompassing the prostate +/- seminal vesicles was used. Patients received 
between 60-66 Gy in 30-33 fractions. Pelvic nodal irradiation was not 
routinely performed. Following treatment patients were followed at 3-6 
monthly intervals with physical examination (including digital rectal 
examination) and PSA determinations. 
Treatment Outcome 
Treatment failure was defined by evidence of biochemical failure (PSA 
rising on two consecutive occasions or PSA &gt;1.5 more than 1 year post 
radiotherapy) and/or evidence of clinical failure (any local progression 
on digital rectal examination or distant recurrence). 
Free PSP94 Determination 
Free PSP94 was measured by competitive ELISA as described in Xuan et al. 
(1996) J. Cell Biochem., 63, 61-73. The sensitivity of the assay was 0.17 
ng. The intra assay variation was &lt;9%, the interassay variation was &lt;10%. 
Archived samples were from the same blood draw used to obtain the 
pretreatment PSA determinations. For the PSP94 assay, aliquots of sera 
were thawed and immediately assayed for PSP94 levels. We had previously 
determined the stability of PSP94 under these conditions by assaying 
aliquots of known PSP94 concentration stored under similar conditions. 
Bound PSP94 Determination 
1 ml of serum was applied to a Sephacryl S-200HR (Pharmacia, Montreal), 
column and eluted by PBS (phosphate buffered saline). About 10 .mu.l of 
sample was taken from fractions just after the major protein (IgG and 
albumin) peaks. Samples were loaded onto a standard 15% SDS-PAGE using 
sample dye containing .beta.-mercaptoethanol and separated by the method 
of Laemmli (Nature (1970), 227, 680-685). Purified human PSP94 was loaded 
as standard at 1, 2, 4 and 8 ng per lane in the same gel. Western blot 
analysis was carried out as in Example 1. Levels of serum bound PSP94 were 
measured according to standard PSP94 density appeared on the same ECL 
(Enhanced chemiluminescence) film. Due to the detection limit of the 
Western blotting plus ECL methods used in this assay, the sensitivity of 
detection of the minimum amount of serum bound PSP94 is .about.50 ng per 
ml. FIG. 6 shows the results of this semi-quantitative assay on patient 
samples with high, median and low (not detectable) levels of serum bound 
PSP94. In 42 of the 44 patients, bound form was determined. There was 
insufficient serum for measurement of bound PSP94 in 2 patients. 
Statistical Analysis 
The following information was retrieved from the clinical records of the 44 
patients: date of diagnosis, clinical stage, pretreatment PSA, tumor 
grade, radiation total dose and number of fractions, date of last 
followup, date of biochemical failure and date of clinical failure. Using 
a statistical software package (STATA, Stata corp,) pretreatment bound and 
free PSP94 levels were correlated with pretreatment PSA, tumor stage and 
grade. Actuarial disease control (as measured from the date of diagnosis) 
as a function of pretreatment PSA, grade, tumor stage and was determined 
by Cox univariate analysis. In addition, actuarial disease control as a 
function of bound and free PSP94 and bound/free PSP94 were determined by 
Cox univariate analysis. 
Serum PSP94 levels in patients 
Of the 42 patients treated, 6 were clinical stage T1, 29 stage T2 and 7 
stage T3. Tumors were well differentiated in 25, moderately differentiated 
in 14, poorly differentiated in 3 and not graded for 2 patients. Median 
pretreatment PSA was 7 ng/ml (range:0.5-93) and median free and bound 
pretreatment PSP94 levels were 10.4 ng/ml (range: 0-79) and 0.49 .mu.g/ml 
(range:&lt;0.05-11.2) respectively. Correlations between PSA; bound PSP94; 
free PSP94, tumor stage and grade are shown in Table 1. Pretreatment PSA 
was loosely correlated with clinical stage but not grade. Pretreatment 
free and bound PSP94 levels were correlated with neither tumor stage, 
grade, PSA, nor each other. 
The 42 patients were treated with radiation to a median dose of 65 Gy 
(range:60-66) and followed for a median of 4.7 years (range: 3.2-5.3 
years) after treatment. To date, 22 patients have failed biochemically. Of 
these 22, 9 also had clinical evidence of recurrent/progressive disease (4 
distant failure, 5 local progression on digital rectal examination). 
Median time to failure was 5.2 years from diagnosis. 
On Cox univariate analysis, pretreatment PSA level was a significant 
predictor of failure post radiotherapy; pretreatment bound PSP94 level 
also correlated with outcome (Table I). In contrast, free PSP94 level was 
not a significant predictor of failure post radiotherapy on univariate 
analysis when analysed as a continuous variable. To detect a possible 
cutoff value of PSP94 as a prognostic indicator, the univariate analysis 
was repeated using the median values of bound and free PSP94 as the cutoff 
as well as by analysing bound and free PSP94 by quartiles. No significant 
cutoff value of bound or free PSP94 was detected in this manner. The 
analysis was also repeated for the subgroups of patients with pretreatment 
PSA less than or greater than the median pretreatment level (7 ng/ml) in 
the group (Table II). Among the subgroups of patients with a favourable 
PSA (&lt;median) pretreatment, bound PSP 94 level (but not PSA level) was a 
significant predictor of relapse survival free survival. For patients with 
an unfavourable PSA (&gt;median) pretreatment, PSA level (but not bound PSP94 
level) was a significant predictor of relapse free survival. On 
multivariate analysis, PSP94 maintained its statistical significance for 
the group as a whole and for the favourable PSA subgroup (Table II). 
TABLE I 
______________________________________ 
Stage 
Grades PSA Bound PSP94 
______________________________________ 
PSA 0.33* -0.03 1 0.03 
Bound PSP94 0.21 -0.02 0.03 1 
Free PSP94 0.03 -0.003 -0.01 0.18 
______________________________________ 
*significant at p = .003 
TABLE II 
______________________________________ 
Univariate 
Multivariate 
Hazard Ratio Hazard Ratio 
Variable Patient Group (p value) (p value) 
______________________________________ 
Stage All * * 
Grade All * * 
PSA All 0.04 (0.000) 0.04 (0.000) 
Bound PSP94 All 0.12 (0.075) 0.17 (0.022) 
Free PSP94 All * * 
Bound PSP94 PSA &lt; 7 0.26 (0.047) 0.26 (0.058) 
(favourable) 
PSA PSA &lt; 7 * * 
(favourable) 
Bound PSP94 PSA &gt; 7 * * 
(unfavourable) 
PSA PSA &gt; 7 0.000 0.03 (0.006) 
(unfavourable) 
______________________________________ 
*Not significant