The present invention describes diagnostic and prognostic assays
to detect in vascular and tissue samples the presence and activity
of the lysosomal pepstatin-insensitive proteinase, CLN2p, which
has been newly found to be associated with breast cancer and serves
as a novel biomarker for breast cancer, including primary, non-primary,
or metastatic breast tumors, neoplasms and carcinomas. The activity
of CLN2p was discovered to be significantly elevated when measured
in breast tissue samples from patients with primary breast carcinoma,
compared with CLN2p levels in normal sample controls, thereby demonstrating
an approximately two- to seventeen-fold higher CLN2p activity in
breast tumors. These higher levels of CLN2p activity in breast tumors
were positively correlated with several known breast cancer biomarkers,
such as cathepsin D, estrogen receptor and progesterone receptor.
The present invention thus provides CLN2p as new biomarker for use
in the detection, diagnosis and prognosis of breast cancer.
What is claimed is:
1. A method of diagnosing breast cancer, comprising (a) contacting
a breast tissue or vascular fluid sample undergoing testing with
an anti-CLN2p antibody under conditions that allow the formation
of a complex between the antibody and CLN2p in the sample; and (b)
detecting the complex; wherein an increased amount of CLN2p in the
sample undergoing testing versus the amount of CLN2p in a normal,
cancer-free control sample diagnoses breast cancer.
2. The method according to claim 1, wherein the CLN2p is contacted
with the antibody in an immunoassay selected from the group consisting
of radioimmunoassay, Western blot assay, immunofluorescent assay,
enzyme immunoassay, immunoprecipitation assay, chemiluminescent
assay, immunohistochemical assay, dot blot assay and slot blot assay.
3. The method according to claim 1, wherein the antibody is a monoclonal
4. The method according to claim 1, wherein the antibody is a polyclonal
5. The method according to claim 2, wherein the immunoassay is
an enzyme linked immunosorbent assay or ELISA.
6. The method according to claim 1, wherein the anti-CLN2p antibody
is detectably labeled.
7. The method according to claim 6, wherein the complex is detected
by a label or indicator selected from the group consisting of immunofluorescent
label, chemiluminescent label, phosphorescent label, enzyme label,
radiolabel, avidin/biotin, colloidal gold particles, colored particles
and magnetic particles.
8. A method of diagnosing breast cancer in a patient, comprising:
(a) contacting a solid matrix having antibodies immunoreactive with
CLN2p immobilized thereto with a patient's breast tissue sample
or a vascular body fluid sample to allow CLN2p in the sample to
bind to the immobilized antibodies; (b) removing unbound sample;
(c) contacting the solid matrix with a labeled antibody specific
for bound CLN2p; and (d) quantifying the amount of CLN2p in the
patient's sample and comparing the amount obtained in (d) with the
CLN2p amount in a normal, non-cancer control, wherein an increased
amount of CLN2p in the patient's sample diagnoses breast cancer
in the patient.
9. The method according to claim 8, wherein the sample is breast
carcinoma, tumor or cancer tissue.
10. The method according to claim 8, wherein the sample is blood,
serum or plasma.
11. The method according to claim 8, wherein the label is selected
from the group consisting of enzyme label, fluorescent label, chemiluminescent
label, phosphorescent label, radioisotope label, colloidal gold,
colored particles, magnetic particles and biotin/avidin.
12. The method according to claim 8, wherein the antibody is polyclonal
13. The method according to claim 8, wherein the amount of CLN2p
in the patient's sample is at least 2-fold greater than the CLN2p
amount in the normal control.
14. A method of diagnosing breast cancer in an individual undergoing
testing, comprising: measuring the level of enzyme activity of CLN2p
in a breast tissue or vascular fluid test sample; and comparing
the CLN2p activity level measured in the test sample with the level
of CLN2p activity in a normal sample control; wherein an increase
in the activity of CLN2p in the test sample relative to the control
sample diagnoses breast cancer in the individual.
15. The method according to claim 14, wherein the vascular fluid
test sample is selected from the group consisting of blood, serum
16. The method according to claim 14, wherein the vascular fluid
test sample is acidified prior to measuring the CLN2p activity.
17. The method according to claim 16, wherein the test sample is
acidified to a pH of between about 3 to about 5.5.
18. The method according to claim 14, wherein the CLN2p activity
in the test sample is at least about 2 to 17-fold greater than the
CLN2p activity in the normal sample control.
19. The method according to claim 14, wherein CLN2p activity is
measured by the amount of enzyme substrate hydrolyzed by the CLN2p
in test samples versus normal control samples.
20. The method according to claim 19, wherein the substrate is
hydrolyzed to Leucine-aminotrifluoromethyl coumarin (L-AFC) product
and the remaining substrate and L-AFC product are detected by high
pressure liquid chromatography.
21. A method of diagnosing breast cancer in a vascular fluid sample
from an individual undergoing testing, comprising: (a) assaying
an aliquot of the vascular fluid sample to determine CLN2p activity;
and (b) comparing the CLN2p activity in the test sample with CLN2p
activity in a normal, noncancerous sample, wherein an increase in
the test sample CLN2p activity relative to the normal sample CLN2p
activity diagnoses breast cancer.
22. The method according to claim 21, wherein the vascular fluid
sample is selected from the group consisting of blood, serum and
23. The method according to claim 22, wherein the vascular fluid
sample is plasma.
24. The method according to claim 21, wherein the CLN2p activity
in the test sample is at least about 2 to 17 fold higher than the
CLN2p activity in the normal sample.
25. A method of diagnosing breast cancer by determining enzyme
levels of CLN2p in a vascular fluid sample from an individual undergoing
testing, comprising: a) acidifying the vascular fluid sample to
a pH of about 3.0 to about 5.5; b) assaying the acidified sample
for CLN2p activity; and c) comparing the CLN2p activity in the individual's
test sample with CLN2p activity in a normal, noncancerous sample,
wherein an increase in CLN2p activity in the test sample relative
to CLN2p activity in the normal sample CLN2p diagnoses breast cancer.
26. The method according to claim 25, wherein the vascular fluid
sample is blood, serum or plasma.
27. The method according to claim 25, wherein the vascular fluid
sample is plasma.
28. An assay method for detecting or diagnosing breast cancer,
comprising: determining CLN2 proteinase levels in a breast tissue
or vascular fluid sample from a patient undergoing testing for cancer,
and comparing the levels of CLN2 proteinase in the patient's sample
with the levels of CLN2 proteinase in a normal sample, wherein increased
levels of CLN2p in the patient's test sample compared with levels
of CLN2 proteinase in the normal sample allows the detection or
diagnosis of breast cancer in the patient.
29. The assay method according to claim 28, wherein the level of
CLN2 proteinase in the patient's test sample is compared with cathepsin
D level; estrogen receptor level; and progesterone receptor level
in the same patient's sample; and further wherein increased levels
of CLN2 proteinase, cathepsin D, estrogen receptor, and/or progesterone
receptor in the test sample compared with CLN2 proteinase, cathepsin
D, estrogen receptor, and/or progesterone receptor levels in a normal
non-cancer sample control allow the diagnosis of breast cancer.
30. The assay method according to claim 28, wherein the vascular
fluid sample is selected from the group consisting of blood, serum
31. The assay method according to claim 30, wherein the vascular
fluid sample is plasma.
32. The method according to claim 28, wherein the level of CLN2
proteinase is detected using high pressure liquid chromatography.
33. The method according to any one of claims 1, 8, 14, 21, 25
and 28, wherein the breast cancer detected or diagnosed is selected
from the group consisting of primary breast cancer, non-primary
breast cancer and metastatic breast cancer.
34. A diagnostic system to detect CLN2p in a biological sample
or vascular fluid, comprising: a) an anti-CLN2p specific antibody
in an amount suitable for at least one assay for detecting the presence
or amount of CLN2p, or fragment thereof, in a sample; b) instructions
for use comprising a description of reagent concentration and/or
at least one assay parameter selected from the group consisting
of: relative amounts of reagent and sample to be admixed, maintenance
time periods for reagent and sample admixtures, temperature and
buffer conditions; and, optionally, c) a detectable label or indicating
agent for signaling the formation of a complex of CLN2p, or bindable
fragment thereof in the sample and the anti-CLN2p antibody.
 This application claims benefit of provisional patent application
U.S. Serial No. 60/188,861, filed Mar. 13, 2000.
FIELD OF THE INVENTION
 The present invention relates to a biomarker newly found
to be associated with breast carcinoma and a sensitive method for
detecting and diagnosing breast cancer in patients.
BACKGROUND OF THE INVENTION
 Lysosomal pepstatin-insensitive proteinase (CLN2p) is a
novel mammalian enzyme discovered as a result of the identification
of a gene defect in the fatal childhood neurodegenerative disorder,
late-infantile neuronal ceroid lipofuscinosis (LINCL) (D. E. Sleat
et al., 1997, "Association of mutations in a lysosomal protein
with classical late-infantile neuronal ceroid lipofuscinosis",
Science, 277:1802-1805). The gene encoding CLN2p is called CLN2.
CLN2p is an abundant lysosomal proteinase with an optimum pH of
3.5 and is extremely stable in frozen tissues or in acidified homogenates
(M. A. Junaid et al., 1999, "A novel assay for lysosomal pepstatin-insensitive
proteinase and its application for the diagnosis of late-infantile
neuronal ceroid lipofuscinosis", Clin. Chim. Acta, 281:169-176).
 Analyses of the substrate cleavage sites have indicated
that CLN2p is the same as tripeptidyl peptidase 1 (EC184.108.40.206) that
cleaves tripeptides from free amino-termini bearing peptides of
varying lengths of between 4-42 residues (D. J. Vines et al., 1999,
"Classical late infantile neuronal ceroid lipofuscinosis fibroblasts
are deficient in lysosomal tripeptidyl peptidase I", FEBS Lett.
443:131-135; M. A. Junaid et al., 2000, "Purification and characterization
of bovine brain lysosomal pepstatin-insensitive proteinase, the
gene product deficient in the human late-infantile neuronal ceroid
lipofuscinosis", J. Neurochem., 74:287-294).
 In carcinoma metastasis, it is believed that proteolytic
enzymes, especially the lysosomal acid proteinases, can mediate
tumor invasion through proteolysis of the basement membrane that
separates the epithelium from the stroma. Hence, a number of laboratories
have investigated the importance of lysosomal enzymes, such as cathepsin
D, as prognostic markers for breast carcinomas (S. M. Thorpe et
al., 1989, "Association between high concentration of Mr 52,000
cathepsin D and poor prognosis in primary human breast cancer",
Cancer Res., 49:6008-6014; A. K. Tandon et al., 1990, "Cathepsin
D and prognosis in breast cancer", N. Engl. J. Med., 322:297-302).
 Some studies have reported that increased levels of cathepsin
D are predictive for breast cancer recurrence (J. A. Foekens et
al., 1993, "Prognostic value of PS2 and cathepsin D in 710
human primary breast tumors: multivariate analysis", J. Clin.
Oncol., 11:899-908). However, other investigators have failed to
confirm this finding (M. Radvin et al., 1994, "Cathepsin D
by Western blotting and immunohistochemistry: failure to confirm
correlations with prognosis in node-negative breast cancer",
J. Clin. Oncol., 12 :467-474). Thus, there is disagreement in the
field regarding the significance and function of cathepsin D and
its relationship to breast cancer, thereby leaving a need for the
discovery of other novel and reliable markers and indicators for
use in the detection, diagnosis, monitoring and/or prognosis of
breast cancer, including breast tumors and carcinomas.
 According to the American Cancer Society (ACS), breast cancer
is the second leading cause of cancer death in women. In 1999 alone,
the ACS estimated that there would be more than 175,000 cases of
invasive breast cancer, resulting in 43,700 deaths. The ACS also
reports that early detection increases survival and treatment options.
Current blood markers are employed for the diagnosis and monitoring
of metastatic breast cancer and not for the early diagnosis of primary
breast cancer. (K. L. Cheung et al., 2000, Cancer Treatment Reviews,
26:91-102). Thus, there is a perpetual need to be able to develop
reliable breast cancer detection and. diagnosis assays for early
detection and involving novel breast cancer biomarkers in an effort
to save lives, ameliorate existing treatments and provide prognostic
indices for breast cancer.
 CLN2p is an acidic lysosomal proteinase that has been found
to be nearly equal in abundance to cathepsin D in brain and other
tissues. For example, cathepsin D activity in human brain (gray
matter) has been reported to be 1069.6.+-.54.4 nmole/hr/mg protein
(D. E. Sleat et al., 1998, Biochem. J., 334:547-551), while human
brain CLN2 proteinase activity has been reported to be 917.+-.43
nmole/hr/mg protein (M. A. Junaid and R. K. Pullarkat, 1999, NeuroSci.
Lett., 264:157-160). In addition, the activity of CLN2p is very
high in tissues/organs that have actively dividing cells, such as
spleen and testes. Due to its abundance, CLN2p may play a crucial
role in metabolism. Table 1 presents exemplary data showing the
distribution and activity of CLN2p in various rat tissues.
1 TABLE 1 Tissue CLN2p Activity (nmol/hr/mg protein) Brain 170
Lung 238 Liver 202 Heart 93 Skeletal Muscle 55 Spleen 1384 Kidney
487 Testis 1039
 In some cancers, proteinases acquire extra significance
due to their purported role in the metastatic process. Much effort
has been expended to exploit the potential of increased proteinases
during metastasis as prognostic markers to predict the disease-free
or overall survival. These studies have relied on semiquantitative
immunoassays to measure the levels of lysosomal acid proteases such
as cathepsin D and cathepsin B (M. K. Schwartz, 1995, "Tissue
cathepsins as tumor markers", Clin. Chim. Acta, 237:67-78).
However, a number of limitations are encountered in the use of semi-quantitative
immunoassays. For example, in the evaluation of estrogen/progesterone
receptors by immunohistochemistry, only the staining of the cell
nuclei is considered positive. Unfortunately, the antigenicity of
receptors may not be conserved during certain procedures used for
fixation. Thus, false negative results are possible.
 The present inventors have newly discovered that the CLN2p
acid proteinase is present and active in tumors from patients with
breast cancer, particularly, primary breast cancer, and that the
amount of CLN2p activity can be correlated with other factors, such
as levels of estrogen receptor, epidermal growth factor receptor,
and progesterone receptor that are known to have prognostic significance
for patients with breast cancer. In addition, the present invention
overcomes the above mentioned limitations of semi-quantitative assays
by employing a quantitative measurement of the enzyme and/or of
the enzyme activity levels of CLN2p in breast cancer, i.e., in breast
cancer and tumor cells.
 The present invention provides the first recognition and
demonstration of CLN2p as a diagnostic and/or prognostic marker
for breast. carcinoma, including breast tumors and cancers, at various
stages of progression. The present invention also advantageously
provides the detection of CLN2p as a blood-based biomarker for breast
cancer through the use of the assays described herein. The assays
and results thereof according to the present invention are specific
for breast cancer, since, in lung cancer, for example, CLN2p activity
is similar to that seen in normal tissues (the average mean CLN2p
activity, .+-.SD, for normal lung and lung tumor tissues are 1418.+-.505
and 1649.+-.200 nmol/hr/mg protein, respectively).
DESCRIPTION OF THE DRAWINGS
 FIGS. 1A and 1B depicts HPLC analyses of the CLN2p activities
in normal (FIG. 1A) and tumor (FIG. 1B) tissues from a patient with
primary breast carcinoma. The substrate G-F-F-L-AFC (280 .mu.M)
was incubated with 7 .mu.g protein in 50 mM ammonium formate buffer,
pH 3.5, containing 0.2 mM pepstatin-A and 0.5 mM E-64 in a final
volume of 25 .mu.l for 5 minutes at 37.degree. C. Reactions were
terminated by adding ice-cold acetone followed by centrifugation
at 12,000.times.g for 2 min. The supernatants were evaporated to
dryness, the dried residue was dissolved in acetonitrile and an
aliquot was analyzed on a reversed-phase C18 HPLC column. The substrate
and the product (L-AFC) were detected by a variable wavelength detector
set at 340 nm.
 FIG. 2 presents the scatter plot of CLN2p enzymatic activity
in breast tissues from normal subjects and in tumors from patients
with primary breast carcinoma. The data show proteinase activity
(solid lines represent mean values) in tumor specimens from 220
patients and in normal tissues obtained from 8 non-cancer subjects
undergoing reductive surgery for macromastia.
SUMMARY OF THE INVENTION
 It is an object of the present invention to provide an enzyme
biomarker that has been newly discovered to be associated with breast
carcinoma and active in breast carcinoma cells, including breast
tumors and breast cancers. In accordance with the present invention,
this enzyme is the pepstatin-insensitive lysosomal tripeptidyl peptidase
CLN2p, which heretofore was disclosed only to be related to the
diagnosis of a rare childhood neurodegenerative disease, late-infantile
neuronal ceroid lipofuscinosis.
 It is another object of the present invention to provide
a method of detection and diagnosis for breast carcinoma, including
breast tumors and cancers, which comprises assaying for the presence
and/or the enzyme activity of CLN2p in breast carcinoma cells, or
in a body tissue or vascular fluid sample, e.g., breast tissue,
tissue extract, cell lysate, blood, serum, or plasma. In accordance
with the present invention, comparative results are obtained by
assaying normal samples, i.e., normal breast tissue, preferably
from a cancer-free individual, other than the breast cancer patient.
Increased CLN2p level or amount, and/or increased level of CLN2p
enzyme activity, in a test sample relative to the values determined
for normal controls can serve to detect and diagnose breast cancer
in a patient. In addition, increased CLN2p activity (i.e., enzyme
activity) in a test sample, relative to CLN2p activity in normal
controls, can also serve in the prognosis of breast cancer in a
patient. Further, the assays for CLN2p levels and activity of the
present invention can be utilized in screening assays, for example,
to screen a patient's breast tissue and blood samples for indications
of cancer. In addition, the assays of the present invention can
be utilized in the early diagnosis of primary breast cancer, as
well as in the detection and diagnosis of non-primary breast cancer
or metastatic breast cancer.
 It is another object of the present invention to provide
antibodies, particularly monoclonal antibodies, that are immunoreactive
with CLN2p, or an immunoreactive portion thereof, and to use such
antibodies in immunoassays, particularly, an enzyme linked immunosorbent
assay (ELISA), to detect and measure CLN2p in breast carcinomas.
 It is yet another object of the present invention to provide
a sensitive and reliable enzyme assay, as well as a reliable and
specific biomarker, for diagnosing breast cancer in an individual,
including the stage of disease exhibited by the individual.
 Yet another object of the present invention is to provide
a sensitive and reliable assay, as well as a reliable and specific
biomarker, for use in the prognosis of the progression of breast
carcinoma in a patient, or for determining or monitoring the outcome
of various treatment or therapy regimens for breast carcinoma in
an individual undergoing such treatment or therapy.
 Another object of the present invention is to provide a
simple assay that can be performed using a vascular body fluid sample,
preferably, a blood, serum, or plasma sample, taken from a patient
or individual being tested for breast carcinoma or undergoing treatment
for breast carcinoma.
 Yet another object of the present invention is to provide
diagnostic and/or prognostic assays in which CLN2p alone, or in
conjunction with an assessment of the status of other cancer-associated
molecules, particularly, cathepsin D levels, estrogen receptor levels,
and/or progesterone receptor levels, that can be used as a diagnostic
and/or prognostic biomarker, or in a panel of biomarkers, for detecting
and monitoring breast carcinomas, including a variety of breast
tumor and cancer types.
 Further objects and advantages afforded by the present invention
will be apparent from the detailed description hereinbelow.
DETAILED DESCRIPTION OF THE INVENTION
 The present invention provides assays and methods involving
the pepstatin-insensitive lysosomal tripeptidyl peptidase CLN2p
as a newly-discovered biomarker for the detection, diagnosis and/or
prognosis of breast carcinoma. Prior to the present invention, CLN2p
was disclosed and considered only to be related to the diagnosis
of a rare childhood neurodegenerative disease--late-infantile neuronal
 The new finding that CLN2p is highly active and has a detectable
activity in a significant number of breast carcinomas tested has
been first discovered by the present inventors. According to the
present invention, CLN2p and its proteinase activity have been found
to be a reliable biomarker for detecting and diagnosing breast cancers,
tumors and carcinomas. Because proteinases can play a prominent
role in tumor progression by facilitating the detachment of tumor
cells from the basement membrane and their spread to other tissues,
the determination of a high level CLN2p and/or high CLN2p activity
in breast tissue or vascular fluid samples is indicative of breast
cancer in a patient.
 That CLN2p activity levels in all but one breast tumor specimen
tested were greater than the highest level observed in normal tissues
(Example 2) indicates that CLN2p is advantageous as a diagnostic
marker for breast cancer. In addition, high CLN2p activity levels
are specific for breast cancer tissue. For example, the activity
of CLN2p in lung cancer tissue was unaffected when compared with
CLN2p activity in normal lung tissue.
 The prognostic significance of CLN2p activity for patients
who are already diagnosed with breast cancer is another aspect of
the present invention which, as is appreciated by the skilled practitioner,
involves direct comparisons to clinical outcomes. Published studies
that have attempted to define a prognostic role of cathepsin D,
another proteinase, in breast cancer have been disappointing. This
may be, in part, due to different forms of cathepsin D, or to a
lack of understanding of the role of cathepsin D in the stromal
components of the tumor, such as infiltrating inflammatory cells
(M. D. Johnson et al., 1993, Cancer Res., 53:873-877). The major
secretory form of cathepsin D in estrogen receptor (ER)-positive
MCF-7 cells is the precursor protein with molecular mass 52 kDa,
which is proteolytically cleaved into the single polypeptide 48
kDa form, and finally processed to the two-subunit mature enzyme
(S. Yonezawa et al., 1988, J. Biol. Chem., 263:6504-16511). While
the 52 kDa form of cathepsin D is inactive, the mature, two subunit
48 kDa form of the enzyme is proteolytically active. By contrast,
CLN2p is active only as a single polypeptide of molecular mass 46
 In further contrast to the findings for cathepsin D (B.
Westley and H. A. Rochefort, 1980, Cell, 20:353-362), the present
inventors have found no difference in the basal secretion or in
the intracellular levels of CLN2p activity, whether or not ER-positive
MCF-7 cells were previously exposed to the hormone .beta.-estradiol.
These results demonstrate that increases in levels of CLN2p and
cathepsin D are controlled by independent and unrelated mechanisms
in breast cancer.
 While a number of earlier studies have related an increase
in cathepsin D following estrogen exposure, there are indications
that the cathepsin D proteinase increases even in some ER-negative
breast carcinoma cell lines as well. Moreover, elevated serum levels
(i.e., 10.+-.8 pmole/ml) of cathepsin D have been reported in 60%
of patients with metastatic breast cancer, while no significant
elevation of cathepsin D was found in women with primary breast
disease, compared with healthy women (i.e., 5.+-.2 pmole/ml and
4.+-.2 pmole/ml, respectively). (J. P. Brouillet et al., 1997, Cancer,
 As agreed by those in the pertinent art (e.g., G. M. Clark,
1994, Breast Cancer Res. Treat., 30:117-126), there is still a strong
need for new and reliable diagnostic and prognostic biomarkers for
breast cancer. In accordance with the present invention, CLN2p has
many desirable properties of a clinically useful biomarker: namely,
it has a high level of activity in nearly all breast tumors; it
has a wide range of activity among different breast tumors; it is
associated with some, but not all, previously established prognostic
factors for breast cancer; the enzyme activity is stable in frozen
samples that have been stored for prolonged periods, and a quantitative
biochemical assay as described herein is available for accurately
determining activity levels of the CLN2p proteinase in samples undergoing
 As described herein, CLN2 proteinase has been found by the
present inventors to have high levels of activity in cells and tissue
specimens from breast cancer patients, including primary breast
cancer patients, and also measurable levels of CLN2p enzyme activity
in plasma samples (the average mean activity, .+-.SD, for normal
samples is 19.9.+-.1.9 nmol/ml/h). In addition, the assays to detect
CLN2p enzymatic activity in samples from breast cancer patients
according to the present invention are sensitive and reliable. For
example, as little as about 2 to 10 .mu.g, preferably 5 to 10 .mu.g,
of tissue protein, or about 5 to 10 .mu.l of plasma sample, is required.
Accordingly, because the proteinase assay requires only about 2
to 10 .mu.g of tissue protein, the diagnostic methods according
to the present invention are well suited for samples that are obtained
by needle biopsy.
 Accordingly, one embodiment of the present invention relates
to CLN2p as an assayable biomarker in cell and tissue samples obtained
from individuals having breast cancer or carcinoma, including various
breast neoplasms, cancers and tumors, and also including primary
disease samples. In this embodiment, the present invention provides
substrate specific enzyme assays that are performed on breast tissue
specimens or samples, e.g. a specimen obtained from a breast biopsy
or aspiration, to determine the enzyme level and activity of the
CLN2p, or CLN2p functional. proteinase fragments, in the specimens
or samples. (Examples 1 and 2).
 Another embodiment of the present invention provides a simple
blood-based assay for breast cancer, including primary breast cancer,
as well as non-primary breast cancers and metastatic breast cancer.
CLN2p has been found to have measurable activity in the normal plasma
samples (19.9.+-.1.9 nmol/ml/h). Moreover, the present invention
has shown that increased CLN2p activity in breast tumors or cancer,
including primary breast tumors and cancer, is readily detected
in view of the high levels of activity of this enzyme that were
newly found in samples of breast cancer tissue. Accordingly, the
increased activity of CLN2p in breast cancer tissue, including primary
breast cancer tissue, can be clinically associated with increased
activity of this biomarker in a vascular fluid sample, such as blood,
serum, or plasma, for assay purposes. The analysis of CLN2p activity
in plasma provides a simple blood based biomarker for breast cancer,
since increased CLN2p activity in the breast tumor may lead to increased
enzyme activity in the plasma. To assay for CLN2p activity in serum
or plasma, such vascular fluid derived from blood samples taken
from an individual to be tested is assayed in a manner similar to
that described for breast cancer tissue or cell samples. (e.g.,
Example 1, Materials and Methods, and Example 4).
 It will be appreciated by those having skill in the art
that vascular fluid, i.e., blood, serum or plasma, has a physiological
(i.e. approximately neutral) pH. Because the CLN2p enzyme is most
stable in an acidic environment, a blood sample to be tested is
thus preferably processed within about an hour or two so as to stabilize
the CLN2p prior to performing the enzyme detection and activity
assays according to the present invention. For processing a blood
sample, the blood sample is collected, preferably in the presence
of an anticoagulant, such as EDTA (K.sub.3EDTA) or heparin. The
sample is centrifuged at low speed to separate the plasma from the
cellular components, and the plasma is immediately frozen at about
-20.degree. C. until use for analysis.
 In another aspect, a blood sample may be acidified immediately
after collection to achieve a pH of about 3.0 to 5.5, preferably
3.5 to 5.0, and more preferably, 3.5 to 4.0, so as to stabilize
the CLN2p. Preferred for lowering the pH of plasma is ammonium formate
buffer, pH 3.5. Those having skill in the art will be aware that
acidifying a blood sample may result in precipitation of serum albumin
in the sample. Thus, removal of the precipitated albumin following
acidification, or otherwise removing the non-precipitated portion
of the blood sample for analysis, is preferably carried out prior
to performing the CLN2p detection or activity assay on an acidified
fresh blood sample according to the present invention.
 In accordance with the present invention, the detection
of increased levels of CLN2p or CLN2p enzymatic activity in a sample
is diagnostic for breast cancer. In normal breast tissues, i.e.,
those obtained from individuals having no detectable breast cancer,
carcinoma or tumor, or no pre-cancerous condition, CLN2p activity
is in the range of from 112 to 343 nmol/hr/mg protein. However,
in breast tumor or cancer tissues, (i.e., from patients having breast
tumors, cancers, or carcinoma), the CLN2p activity is generally
in the range of from 496 to 5787 nmol/hr/mg protein.
 Comparatively, the CLN2p activity in breast tumor or cancer
tissues is at least about 2 to 17 fold higher than the CLN2p activity
in normal tissues. Accordingly, higher CLN2p activity in breast
tissue is indicative of cancer and is directly associated with a
cancerous condition. The amount or level of CLN2p in the breast
tumor or cancer tissue is generally proportional to the increased
activity level of the enzyme detected in the tissue. Thus, the amount
or level of CLN2p is expected to be at least about 2 to 17 fold
higher in breast cancer versus normal, non-cancer tissue. The normal
CL2Np activity values represent those determined from tissues that
have been obtained from cancer-free individuals at the time of reductive
surgical procedures, rather than from tissues that appear to be
normal in breast tumor or cancer patients. Such normal tissue controls
were employed because apparently "normal" appearing tissues
from patients with breast cancer were often found to have higher
CLN2p activity. Thus, the changes in breast cancer patients can
be detected by increased CLN2p activity according to this invention,
even though such differences may not be detectable by means of histochemistry.
 Monitoring changes in the level of the CLN2p may facilitate
the diagnosis and detection of breast cancer, thereby allowing the
determination of a course of treatment or therapy, or assessing
outcome of a treatment or therapy for a breast cancer patient. Thus,
by monitoring the level of CLN2p, an abnormal level of the enzyme,
or an increase in the enzyme activity, can be indicative of a cancerous
condition that can be reproducibly detected so that treatment protocols
can be appropriately determined and carried out. In addition, the
detection of higher levels, or sustained high levels, of CLN2p activity
in a breast cancer patient, compared with normal CLN2p values may
serve to indicate a poor prognosis and/or an increased chance of
recurrence of breast cancer.
 Another embodiment of the present invention relates to antibodies
that are immunoreactive with the CLN2p enzyme, or immunogenic fragments
or portions thereof, particularly for use in antibody-based assays
(e.g., ELISAs) for detecting CLN2p in cell or tissue samples obtained
from a breast carcinoma, tumor or cancer, or in a patient's vascular
fluid sample. Both polyclonal antibodies (PAbs) and monoclonal antibodies
(MAbs) specific for CLN2p, or an immunogenic fragment or portion
thereof, are encompassed by the present invention. CLN2p isolated
and/or purified from natural sources (e.g., M. A. Junaid et al.,
2000, J. Neurochemistry, 74:287-294), or synthetically or recombinantly-produced
CLN2p enzyme, or peptides thereof, may be used as an immunogen for
injecting into a host animal to elicit a specific immune response
and to generate anti-CLN2p antibodies. Immunogenic fragments or
portions of CLN2p may be obtained by the use of proteolytic enzymes,
or by the synthesis of peptides bearing epitopes that elicit an
immune response and generate antibodies, using methods and protocols
practiced in the art.
 In another embodiment, the present invention contemplates
a method of producing an antibody that is immunoreactive with CLN2p,
or an immunoreactive fragment or portion thereof, comprising: immunizing
an animal with CLN2p as described above as an immunogen, isolating
serum from the animal, wherein the serum contains antibodies that
are immunoreactive with epitopes on the CLN2p immunogen and further
purifying the anti-CLN2p antibodies, if desired, using immunoglobulin
purification techniques practiced in the art. Alternatively, the
spleen of an immunized animal, preferably a mouse, can be used in
conventionally practiced hybridoma production techniques, as described
herein, and monoclonal anti-CLN2p antibodies can be produced and,
if desired, isolated for use.
 As used herein, "antibody" or "antibodies"
refers to intact molecules as well as fragments thereof, such as
Fab, F(ab).sub.2, and Fv, which are capable of binding an epitopic
determinant of the immunogen CLN2p. As will be appreciated by those
having skill in the arti the immunogen can be conjugated to a carrier
protein, if desired, to increase immunogenicity, particularly, if
a small peptide or CLN2p fragment is used. Commonly used carriers
that are routinely chemically coupled to peptides include serum
albumins, i.e., bovine, sheep, goat, or fish serum albumin; thyroglobulin;
and keyhole limpet hemocyanin. The coupled immunogen-carrier is
then used to immunize a recipient animal (e.g., mouse, rat, sheep,
goat, or rabbit).
 The term "antigenic determinant" refers to that
fragment of a molecule (i.e., an epitope) that makes contact with
a particular antibody. When an isolated and/or purified CLN2p is
used to immunize a host animal, numerous regions of the enzyme may
induce the production of antibodies which bind specifically to a
given region or three-dimensional structure on the enzyme; these
regions or structures are referred to as antigenic determinants
or epitopes. An antigenic determinant may compete with the intact
antigen (i.e., the immunogen used to elicit the immune response)
for binding to an antibody.
 The antibodies can be elicited in an animal host by immunization
with CLN2p-derived immunogenic components, or can be formed by in
vitro immunization (sensitization) of immune cells. The antibodies
can also be produced in recombinant systems transformed, transfected,
infected or transduced with appropriate antibody-encoding DNA. Alternatively,
the antibodies can be constructed by biochemical reconstitution
of purified heavy and light chains. Antibodies embraced by the present
invention include hybrid antibodies, chimeric antibodies, humanized
antibodies (see, for example, U.S. Pat. No. 5,585,089 to C. J. Queen
et al.) and univalent antibodies. Using such antibodies, for example,
CLN2p, or an immunogenic fragment or portion thereof, can be detected
in a test sample by chromatography on antibody-conjugated solid-phase
matrices or supports (see E. Harlow and D. Lane, 1999, Using Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.), or by immunoassay. Preferred are antibodies that
specifically recognize and bind to CLN2p, and do not recognize and
bind to similar enzymes or proteins.
 The antibodies can be used as an in vitro diagnostic agent
to test (or screen) for the presence of CLN2p in biological samples,
particularly, breast tissue samples and vascular fluid samples,
in standard immunoassay protocols. Preferably, the assays which
use the antibodies to. detect the presence of CLN2p in a sample
involve contacting the sample with at least one of the antibodies
under conditions which will allow the formation of an immunological
complex between the antibody and the CLN2p antigen that may be present
in the sample. The formation of an immunological complex if any,
indicating the presence of CLN2p in the sample, is then detected
and measured by suitable means.
 The assays embraced by the present invention may be, for
example, of direct format (where the labeled first antibody reacts
with the antigen, e.g., CLN2p), an indirect format (where a labeled
second antibody reacts with the first antibody that binds to the
antigen), a competitive format (such as the addition of a labeled
antigen), or a sandwich format (where both labeled and unlabelled
antibodies are utilized), as well as other formats described in
the art. In one such assay, the biological sample is contacted with
antibodies of the present invention and a labeled second antibody
is used to detect the presence of CLN2p, to which the antibodies
are bound and a complex is formed between the antibody and bound
 Such assays include, but are not limited to, radioimmunoassays
(RIA), ELISA, indirect immunofluorescence assays, Western blot assays,
immunohistochemical assays, chemiluminescent assays, immunoprecipitation
assays, dot blot assays, slot blot assays and the like. The antibodies
may be labeled or unlabeled depending on the type of assay used.
Labels which may be coupled to the antibodies include those known
in the art and include, but are not limited to, enzymes (e.g., horseradish
peroxidase or glucose oxidase), radioisotopes, fluorogenic (e.g.,
fluorescein isothiocyanate (FITC), fluorescein isocyanate (FIC),
5-dimethylamine-1-napthalenesulfonyl chloride (DANSC), tetramethyl-rhodamine
isothiocyanate (TRITC), lissamine, and the like), chromogenic substrates,
cofactors, biotin/avidin, chemiluminescent compounds, phosphorescent
compounds, colloidal gold, colored particles and magnetc particles.
 As is appreciated by the skilled practitioner, in such cases
in which the principal indicating group is an enzyme, additional
reagents (e.g., substrates) are required for the production of a
visible signal. Radioactive elements of various classes, such as
.sup.124I, .sup.125I, .sup.131I, .sup.51Cr, (gamma ray emitters);
.sup.32P, .sup.3H, 35S (beta emitters), and .sup.11C, .sup.14C,
.sup.15O, or .sup.13N (positron emitters), may also be used as detectable
labels. The labeled complex may be detected visually, with a spectrophotometer,
or by another detector, depending on the labeling or indicating
 Modification of the antibodies allows for coupling by any
known means to carrier proteins or peptides or to known water-insoluble
supports or matrices, for example, polystyrene or polyvinylchloride
microtiter plates, wells, or tubes; glass tubes or glass beads;
and chromatographic supports, such as paper, cellulose and cellulose
derivatives, and silica. Other suitable solid supports or matrices
include the following as nonlimiting examples: crosslinked dextran
(Pharmacia, Piscataway, N.J.); agarose, polystyrene beads (about
1-5 microns in diameter; e.g., Abbott Laboratories, Illinois); and
crosslinked polyacrylamide; nitrocellulose- or nylon-based webs,
such as sheets, strips, paddles, or sticks.
 One method for utilizing the antibodies according to the
present invention to detect CLN2p in a breast cancer-related sample
comprises an immunoassay which utilizes a solid support or matrix
to which are bound antibodies that recognize all or a portion of
the CLN2p, contacting the support with the sample or an aliquot
of the sample and detecting the CLN2p via a radioactive or non-radioactively
labeled detection molecule (i.e., an appropriate antibody) that
specifically binds to the CLN2p which is bound to the anti-CLN2p
antibodies adsorbed onto the solid support or matrix.
 Another method for detecting CLN2p in a breast cancer-related
sample comprises incubating the sample with antibodies that specifically
recognize and bind to CLN2p, or an immunogenic fragment or portion
thereof, under conditions that allow the antibodies to bind to the
enzyme, and then determining the binding of the antibodies to the
CLN2p, for example, by adding detectable antibodies that bind to
a different epitope on the CLN2p and are coupled to a solid support,
or by adding antibodies that bind to the antibodies already bound
to CLN2p, with the added antibodies being labeled and providing
a selectable marker. The added selectable antibodies, or bindable
fragments thereof, may be bound to a solid support, or they many,
in turn, be bound by other detectable antibodies which are bound
to a support. Such immunoassay methods and variations thereof are
known and practiced in the art.
 As a preferred, but nonlimiting example, the present invention
provides the ELISA format (Engvall et al., 1971, Immunochemistry,
8:871-4; Basic and Clinical Immunochemistry, Chapt. 22, 4th Edition,
D. P. Stites et al., Lange Medical Publications, Los Altos, Calif.,
1982; and D. J. Reen, 1994, Methods Mol. Biol. 32:461-6) as a specifically
useful and practical antibody-based method to detect, diagnose,
and/or quantify the presence of CLN2p in the breast tissue or fluid
sample of an individual undergoing testing either in vivo or in
vitro. A variety of antibody-based ELISA protocols are known and
practiced in the art and are suitable for use in the present invention.
 The present invention thus embraces a diagnostic system,
particularly diagnostic kits, that are used in the ELISA format
to detect CLN2p in a biological tissue/cell sample or vascular fluid.
For example, a suitable diagnostic system includes, in an amount
suitable for at least one assay, an anti-CLN2p specific antibody
as a separately packaged immunological reagent for assaying for
the presence or amount of CLN2p, or fragment thereof, in a sample.
Instructions for use of the packaged reagent are also typically
included. Instructions for use generally include a description of
the reagent concentration, or at least one assay method parameter,
such as the relative amounts of reagent and sample to be admixed,
maintenance time periods for reagent/sample admixtures, temperature,
buffer conditions, and the like. A preferred diagnostic system further
includes a detectable label or indicating agent for signaling the
formation of a complex of, for example, a CLN2p or bindable fragment
thereof, and an anti-CLN2p antibody according to the present invention.
 In the immunoassays for use in the present invention, an
anti-CLN2p antibody, monoclonal or polyclonal, serves as an immunochemical
reagent to form an immunoreaction product whose amount relates,
either directly or indirectly, to the amount of CLN2p in the sample.
Also contemplated are immunoassay methods for determining the amount
of CLN2p, or a detectable fragment thereof, in a biological fluid
sample using a CLN2p, or polypeptide fragment thereof, as a reagent
to form a product whose amount relates, either directly or indirectly,
to the amount of CLN2p in the sample. Such assays embrace detection,
diagnostic, screening and prognostic assays.
 Hybridomas that produce monoclonal antibodies against the
immunogenic components of CLN2p can be produced by well-known techniques.
Hybridomas can be produced by the fusion of an immortalized cell
line with a B-lymphocyte that produces the desired antibody. Alternatively,
non-fusion techniques for generating immortal antibody-producing
cell lines are possible, and are within the purview of the present
invention (see Casali et al., 1986, Science, 234:476). Immortalized
cell lines are typically transformed mammalian cells, particularly
myeloma cells of rodent, bovine, or human origin. Most frequently,
rat or mouse myeloma cell lines are employed as a matter of convenience
 Standard procedures can be used to select hybridomas, such
as HAT (hypoxanthine-aminopterin-thymidine) selection. Hybridomas
that secrete desired monoclonal antibodies can be selected by assaying
the cells' culture medium by standard immunoassays, such as immunoblotting,
ELISA, radioimmunoassay (RIA), or comparable assays. Antibodies
which immunoreact with CLN2p can be recovered and isolated from
the medium using standard protein purification techniques (see Tijssen,
1985, Practice and Theory of Enzyme Immunoassays, Elsevier, Amsterdam).
 In another embodiment, the present invention encompasses
an assay to detect CLN2p enzyme activity levels in a tissue or vascular
fluid (e.g., plasma or serum) sample undergoing testing. For breast
tissue samples, such assays involve the preparation of tissue extracts
and homogenates using procedures practiced in the relevant art,
and the supernatants therefrom. The level of CLN2p activity is measured
by determining the amount of substrate hydrolysis (i.e., the amount
of hydrolysis of CBZ-Arg-Gly-Phe-Phe-Leu-AFC peptide substrate to
the product Leu-AFC, as described in Examples 1 and 2) that is generated
by the CLN2p in the test sample. The test sample results are compared
with enzyme activity levels from normal samples. High pressure liquid
chromatography (HPLC) as described in Examples 1 and 2 is employed
to quantify the results of the CLN2p activity analysis.
 Another embodiment of the present invention relates to detection,
diagnostic, or prognostic assays in which CLN2p alone, or in conjunction
with an assessment of the status of other cancer-associated molecules,
particularly, cathepsin D levels, estrogen receptor (ER) levels,
and/or progesterone receptor (PgR) levels, can be used as diagnostic
and/or prognostic biomarker(s) for detecting and monitoring breast
carcinomas, including a variety of breast tumor and cancer types.
CLN2p levels, assayed in conjunction with a panel of cathepsin D,
ER and PgR levels, provide a diagnostic, or a prognostic, biomarker
assay for breast carcinoma and possible disease outcome.
 Other binding assays that are well known to the person of
ordinary skill in the art are encompassed by the present invention
for detecting CLN2p in a sample. Such assays include the antibody
based immunoassays described above and further include numerous
other binding assays, for example, in which a target protein (e.g.,
CLN2p) in a sample may be reacted with a binding molecule capable
of specifically binding to the target protein. The binding molecule
may comprise, for example, a member of a ligand-receptor pair (i.e.,
a pair of molecules capable of specific binding interactions), antibody-antigen,
enzyme-substrate, nucleic acid-nucleic acid, protein-nucleic acid,
or other specific binding pairs and members thereof as known in
the art. Binding molecules may be designed so that they have enhanced
affinity for the target protein. Binding molecules may be linked
or coupled to a detectable label or indicator for detection by means
known in the art. Means for coupling labels or indicators to binding
molecules are well known in the art; commercial kits for labeling
proteins and the like, are also available.
 The following examples as set forth herein are meant to
illustrate and exemplify the various aspects of carrying out the
present invention and are not intended to limit the invention in
 Materials and Methods
 Human breast carcinoma tissues were obtained either from
the National Breast Cancer Tissue Resource, Baylor College of Medicine,
Houston, Tex., or from the National Cancer Institute Cooperative
Human Tissue Network, Philadelphia, Pa. Tumor specimens from 220
patients with primary breast carcinoma, and normal tissues from
8 non-cancer subjects undergoing reductive surgery for macromastia,
without any known signs of neoplasia or pre-cancerous condition,
were used in the studies described herein. The results of the CLN2p
activity in breast tumors were compared with CLN2p activity from
normal breast tissue obtained as described above. "Normal-appearing"
breast tissue from breast cancer patients, or from the patient whose
cancerous sample is undergoing testing, are not suitable for obtaining
a normal reference value, since such "normal-appearing"
tissues generally had higher CLN2p activity. In contrast, unlike
the significantly higher level of CLN2p activity in tumor versus
normal breast tissue, the CLN2p activity in normal lung tissue was
similar to the CLN2p activity detected in cancerous lung tissue.
 To measure CLN2p activity in tissue sources, tissues (50-100
mg) were homogenized in 2 ml of 50 mM ammonium formate buffer, pH
3.5, containing 0.15 M NaCl and 0.1% Triton X-100 in a variable
speed tissue tearor. The supernatants obtained after centrifugation
at 14,000.times.g for 10 minutes were used for CLN2p determinations.
The extracts were stored frozen at -20.degree. C. if not used immediately.
 Substrate Preparation
 The preparation of the tetrapeptide substrate GFFL-7-amino4-trifluoromethyl
coumarin (>99% pure) and measurement of the CLN2p activity in
tissue extracts by HPLC were virtually as described by M. A. Junaid
et al., 1999, Clin. Chim. Acta, 281:169-176; M. A. Junaid et al.,
2000, J. Neurochem., 74:287-294; and M. A. Junaid et al., 1999,
Neurosci. Lett., 264:157-160.
 Synthetic peptide CBZ-Arg-Gly-Phe-Phe-Leu-aminotrifluoromethyl
coumarin (AFC) was purchased from Enzyme Systems, Products, Livermore,
Calif. All other chemicals were from Sigma Chemical Co., St. Louis,
Mo. Reversed-phase C18 high pressure liquid chromatography (HPLC)
column was purchased from Rainin Instruments Co., Woburn, Wash.
 The peptide CBZ-Arg-Gly-Phe-Phe-Leu-AFC (2 mg) in 0.25 mL
50% acetonitrile was treated with 100 .mu.g Trypsin in 100 mM Tris-HCl,
pH 7.4, at room temperature until completion of the reaction. The
progress of the reaction was ascertained by analyzing aliquots of
the reaction mixture by reversed-phase HPLC. The product Gly-Phe-Phe-Leu-AFC
that was formed was purified by reversed-phase HPLC and evaporated
under nitrogen to dryness (>99% pure). These HPLC conditions
are the same as described under HPLC analysis.
 CLN2p Assay
 An aliquot of the supernatant containing 5-10 .mu.g protein
was incubated with 7 nmol (280 .mu.M) of the peptide substrate Gly-Phe-Phe-Leu-AFC
(dissolved in 40% dimethyl formamide) in 20 mM ammonium formate
buffer pH 3.5, containing 0.2 mM pepstatin-A and 0.5 mM E-64. The
final volume of the assay mixture was 25 .mu.L. Incubations were
carried out at 37.degree. C. for 5-10 min., after which the reaction
was terminated by the addition of 100 .mu.L ice-cold acetone. The
supernatant obtained after centrifugation at 12,000.times.g for
2 min. was dried under nitrogen, resuspended in 50 .mu.L of 60%
acetonitrile, and 10 .mu.L was used for HPLC analysis.
 HPLC Analysis
 The reaction products were analyzed by reversed-phase HPLC
on a Microsorb-MV C18 column (0.46.times.10 cm, 3 .mu.m, 100 .ANG.)
in an isocratic solvent system comprised of 55% acetonitrile and
0.1% trifluoroacetic acid at a flow rate of 0.5 mL/min. Eluates
were detected by a variable wavelength ultraviolet (UV) detector
at 340 nm, and the peaks were integrated using a Spectra-Physics
SP 4290 integrator. The detector full-range and the chart speed
were set at 0.001 and 0.5 cm/min., respectively. CLN2p activity
was calculated based upon the amount of Leu-AFC formed from the
area of the corresponding peaks obtained by injecting known amounts
 Protein concentrations in the extracts were determined by
a modified method of Lowry et al. (O. H. Lowry et al., 1951, J.
Biol. Chem., 193:265-275) using bovine serum albumin as the standard.
CLN2p activity in the extracts was expressed as nmol/h/mg protein
and was correlated with estrogen receptor (ER), progesterone receptor
(PgR) and epidermal growth factor receptor (EGFR) levels, cathepsin
D activity, S-phase fraction and DNA ploidy status.
 The standard multipoint, dextran-coated charcoal assay was
modified as previously described to incorporate .sup.125I-labeled
estradiol and [.sup.3H]R5020 in a single assay, allowing the simultaneous
determination of both ER and PgR (L. G. Dressler et al., 1988, Cancer,
61:420-427). Levels greater than or equal to 3 fmol/mg protein were
considered to be positive for ER, and levels greater than or equal
to 5 fmol/mg protein were considered to be positive for PgR.
 EGFR was measured by a radioligand binding assay, using
fixed concentrations of radiolabeled EGF and varying concentrations
of unlabeled EGF. Malignant tissue was pulverized and homogenized
prior to ultracentrifugation at approximately 108,000.times.g for
1 hour at 4.degree. C. Following removal of the fat and cytosol,
the membrane pellet was homogenized again and briefly centrifuged.
The sample was added to labeled EGF and incubated. The separation
of bound from free EGF was accomplished with a polyethyleneglycol
gradient, and the bound fraction was quantified on a gamma counter.
Following subtraction of nonspecific binding, the data were analyzed
via Scatchard analysis and reported in fmol/mg of membrane protein.
Levels greater than or equal to 10 fmol/mg protein were considered
to be positive.
 An immunoradiometric (IRMA) variant of the double antibody
technique was used to measure cathepsin D levels in tumor cytosols.
Cytosols were prepared in 10 mM Tris buffer containing 1.5 mM EDTA,
5 mM dithiothreitol and 10 mM molybdate. Cytosol protein concentration
was adjusted to approximately 1-2 mg/mL prior to a 60-fold dilution.
Three reference cytosols prepared from pooled disaggregated breast
tumor tissue were included in each assay, together with a control
provided by the manufacturer. The immunoradiometric assay (CIS-US,
Inc., Bedford, Mass.) employed the D7E3 monoclonal antibody as the
capture antibody and the radiolabeled M1G8 monoclonal antibody as
the detection antibody. Bound radioactivity was directly proportional
to the level of cathepsin D in the specimen. Cathepsin levels greater
than 50 pmol/mg cytosol protein were considered high.
 DNA analyses were performed on breast tumor or cancer cells.
DNA ploidy and S-phase fraction were determined by flow cytometry
as described previously (L. G. Dressler et al., 1988, Cancer, 61:420-427
and C. R. Wenger et al., 1993, Breast Cancer Res. Treat, 28:9-20).
Ploidy refers to the ratio of the amount of DNA in a tumor to the
amount of DNA in normal diploid cells. S-phase by flow cytometry
measures all cells that are actively synthesizing DNA. S-phase fractions
were estimated using the MODFIT program (Verity Software House,
Inc., Topsham, Me.), with single-cut debris stripping and single
trapezoids for the S-phase components. S-phase fractions greater
than or equal to 6.7 for diploid tumors and greater than or equal
to 11.0 for aneuploid tumors were considered high.
 Statistical Analyses
 CLN2p activities in normal and breast carcinoma tissues
were expressed as mean .+-.SD and compared by a two-sample Student's
t test at the 5% level of significance. Associations between CLN2p
activity and other biomarkers for breast carcinoma were described
by Spearman rank correlations (r.sub.s) or Wilcoxon rank sum tests.
 HPLC analyses of CLN2p activity in breast tumor tissue from
a patient with breast carcinoma and in normal non-cancerous breast
tissue were performed. The results are shown in FIGS. 1A and 1B.
In the normal tissue, less than 5% of the substrate was hydrolyzed
to L-AFC, while over 90% of the substrate was hydrolyzed in the
cancer tissue, when the same amount of protein was incubated. In
the patient samples presented in FIGS. 1A and 1B, these values correspond
to CLN2p specific activity of about 300 and 3000 nmol/h/mg protein
for the normal and the tumor tissues, respectively.
 FIG. 2 shows the scatter plot of CLN2p activity in breast
tissues from normal subjects and in tumors from patients with primary
breast carcinoma. The levels of CLN2p ranged from 112-343 nmol/h/mg
protein for normal tissues (n=8) and 164-5787 nmol/h/mg protein
for tumor tissues (n=220).
 The results indicate that breast tumors have at least a
seven-fold higher CLN2p activity (1813.+-.834, mean .+-.S.D. P<0.001)
when compared with tissues from normal subjects (251.+-.66, mean
.+-.S.D.). All but 1 sample out of the total 220 tumor tissues analyzed
had CLN2p activity greater than the highest normal value determined.
This suggests that CLN2p activity is a clinically useful diagnostic
test for breast carcinoma. It is not known why one of the tumor
tissues possessed such low CLN2p activity. Interestingly, this one
particular sample (with CLN2p activity of 164 nmol/hr/mg protein)
appeared to be aberrant in other ways in that it also had a lower
cathepsin D value, negative ER, PgR and EGFR levels, diploid DNA
ploidy status and low S-phase fraction. Without wishing to be bound
by theory, or limited by one atypical sample, simple biological
variation or mutation may be attributed to the aberrant values obtained
for the single anomalous patient sample showing a low CLN2p value.
 CLN2p activity was compared with other therapeutic biomarkers
measured in the breast cancer specimens (see Example 1). Associations
with these factors expressed as dichotomous variables are shown
in Table 2.
 Test descriptions for Estrogen Receptor (ER)/Progesterone
Receptor (PgR) indicate that the presence and levels of hormone
receptors in tumor cells (e.g., breast tumor cells) are strongly
correlated with response to hormonal therapy and clinical outcome.
(H. Battifora, 1994 Appl. Immunohistochem., 2(3):143-145; S. M.
Veronese et al., 1995, Appl. Immunohistochem., b3:85-90; I. deMascarel
et al., 1995, Appl. Immunohistochem., 3:222-231; L. P. Pertshuk
et al., 1996, Cancer, 77:2514-2519; N. Weidner, 1996, Calif. Seminars
in Pathology, 10-14; C. Cohen et al., 1997, Abstr. Annual Meeting
of the USCAP, 181A; C. T. Taylor, 1996, Cancer, 77(12):2419-2422).
The overexpression of Epidermal Growth Factor Receptor (EGFR) is
associated with poor prognosis. (N. Weidner et al., 1994, Breast
Cancer Res. Treatment, 29:97-107; J. Baselga et al., 1994, Breast
Cancer Res. Treatment, 29:127-138).
2TABLE 2 Comparison of CLN2p activity with different levels of
other biomarkers in 200 samples from patients with primary breast
carcinoma CLN2p activity nmol/hr/mg protein Wilcoxon Marker Status
Number (Mean .+-. S.D.) p-value Cathepsin D low 133 1705 .+-. 784
<0.0001 high 67 2157 .+-. 824 Estrogen receptor - 39 1400 .+-.
594 <0.0001 (ER) + 161 1967 .+-. 835 Progesterone - 90 1516 .+-.
586 <0.0001 receptor (PgR) + 110 2135 .+-. 885 Epidermal Growth
- 154 1917 .+-. 876 0.16 Factor Receptor + 46 1654 .+-. 583 (EGFR)
DNA ploidy Diploid 97 1839 .+-. 873 0.41 Aneuploid 103 1874 .+-.
778 S-phase fraction Low 117 1918 .+-. 886 0.33 High 83 1769 .+-.
 CLN2p activity was positively correlated with cathepsin
D, ER and PgR (P<0.0001). No correlations, either positive or
negative, were observed with EGFR, DNA ploidy status or S-phase
fraction. Similar results were observed when the prognostic biomarker
measurements were expressed as continuous variables (Table 3).
3TABLE 3 Comparison of CLN2p activity with other biomarkers in
200 samples from patients with primary breast carcinoma Correlation
Values coefficient, Variable (Mean .+-. S.D.) (r.sub.s) p-value
Cathepsin D 47.4 .+-. 32.5 0.45 0.0001 Estrogen receptor 121 .+-.
165 0.31 0.0001 Progesterone 191 .+-. 319 0.43 0.0001 receptor EGFR
33.1 .+-. 212 -0.11 0.12 S-Phase fraction 9.1 .+-. 6.3 -0.04 0.58
 As seen from Table 3 above, CLN2p activity was significantly
correlated with cathepsin D (r.sub.s=0.45, p<0.0001), as determined
by immunohistochemical staining of cytoplasm and stroma of tumor
cells; ER (r.sub.s=0.31, p<0.0001) and PgR levels (r.sub.s=0.43,
p<0.0001), as determined by immuno-histochemical staining of
tumor cell nuclei. However, no significant correlations were observed
with EGFR levels or with S-phase fraction. Multivariate regression
analyses, using both continuous or dichotomous representations,
revealed that cathepsin D and PgR were the strongest predictors
for CLN2p activity in the breast cancer tissues (r.sup.2=0.24 and
0.20, respectively). Collectively, the results from Example 3 suggest
that CLN2p alone, or in conjunction with cathepsin D, ER and/or
PgR status, can be an attractive prognostic biomarker assay for
breast carcinomas, including breast cancers and tumors.
 This example demonstrates that an analysis of the levels
of CLN2p activity, cathepsin D activity, and ER and/or PgR levels
may be employed as reliable biomarkers in a panel of biomarkers
for the detection and/or prognosis of breast cancer to provide a
reliable indication of a patient's breast cancer disease or condition
 This example describes the use of an assay method according
to the present invention to determine CLN2p in blood samples, particularly,
 For plasma samples, freshly collected heparinized blood
was centrifuged at low speed (.about.1000.times.g) to separate the
plasma component from the cellular component. The plasma was stored
frozen at -20.degree. C. if it was not used immediately for analysis.
Prior to carrying out the assay, the plasma samples were centrifuged
at 12,000.times.g for 5 minutes to remove any contaminating platelets.
For CLN2p activity determinations, 5 .mu.l of plasma was incubated
with 280 .mu.M of the peptide substrate Gly-Phe-Phe-Leu-AFC in 20
mM ammonium formate buffer, pH 3.5 containing 0.2 mM pepstatin-A
and 0.5 mM E-64 (inhibitor of enzymes having active thiol groups,
Sigma). The final volume of the assay mixture was 25 .mu.l. Incubations
were carried out at 37.degree. C. for 10 minutes, after which the
reaction was terminated by the addition of 100 .mu.l of ice-cold
acetone. The supernatant obtained after centrifugation at 12,000.times.g
for 2 minutes was dried under nitrogen, resuspended in 50 .mu.l
of 60% acetonitrile, and 25 .mu.l was used for HPLC analysis as
described in Example 1.
 The results from 8 normal plasma samples obtained using
the above protocol showed that CLN2p activity in these samples was
19.9.+-.1.9 nmol/hr/ml plasma. A plasma sample obtained from a breast
cancer patient using the protocol above and assayed for CL2Np activity
as described herein showed CLN2p activity that was about 10-fold
higher than that of the normal plasma controls as presented in Table
4. Thus, plasma provides a suitable vascular fluid source from which
to obtain CLN2p activity data and results.
4TABLE 4 CLN2 proteinase activities in plasma Proteinase Activity
Sample (nmol/hr/ml) Normal 19.9 .+-. 1.9* (17.0-22.3) Primary breast
cancer 190.8 .+-. 81.8 (138-293.6) *Mean .+-. standard deviation
 The contents of all patents, patent applications, published
articles, books, reference manuals and abstracts cited herein are
hereby incorporated by reference in their entirety to more fully
describe the state of the art to which the invention pertains.
 As various changes can be made in the above-described subject
matter without departing from the scope and spirit of the present
invention, it is intended that all subject matter contained in the
above description, or defined in the appended claims, be interpreted
as descriptive and illustrative of the present invention. Many modifications
and variations of the present invention are possible in light of
the above teachings.