Human Glandular Kallikrein in Breast Milk, Amniotic Fluid, and Breast Cyst Fluid

Clinical Chemistry 45:10
1774 –1780 (1999)
Enzymes and Protein
Markers
Human Glandular Kallikrein in Breast Milk,
Amniotic Fluid, and Breast Cyst Fluid
Angeliki Magklara,1 Andreas Scorilas,1 Carlos Lo´pez-Ot?´n,2 Francisco Vizoso,3
Alvaro Ruibal,4 and Eleftherios P. Diamandis1*
Background: Human glandular kallikrein (hK2) be-
Conclusions: The female breast produces hK2 in addi-
longs to the serine protease family of enzymes and has
tion to PSA. More studies are necessary to establish the
high sequence homology with prostate-specific antigen
role of this kallikrein in nondiseased breast, gross
(PSA). The physiological role of hK2 has not as yet been
breast cystic disease, and breast cancer.
determined, but there is evidence that it can regulate the
© 1999 American Association for Clinical Chemistry
proteolytic activity of PSA through processing and acti-
vating pro-PSA, an inactive precursor. Thus, it is con-
The human kallikrein gene family is a subgroup of a large
ceivable that these two secreted proteins may coexist in
family of serine proteases and currently includes three
biological fluids. Currently, hK2 is considered an andro-
members: tissue kallikrein, human glandular kallikrein
gen-regulated and prostate-specific protein. Recently, it
(hK2),5 and prostate-specific antigen (PSA or hK3) (1, 2 ).
has been demonstrated that hK2 is expressed in the
PSA is a 33-kDa protease with chymotrypsin-like activity
breast cancer cell line T-47D after stimulation by steroid
(3 ) and was, until recently, thought to be produced
hormones, and we reported that hK2 can be detected in
exclusively by epithelial cells of the prostate gland (4, 5 ).
a subset of breast tumor extracts. These data suggest that
PSA is the most reliable tumor marker available, and it is
hK2 may be expressed in tissues other than the prostate,
widely used for prostate cancer diagnosis and manage-
such as those in which PSA has already been detected.
ment (6 ). It is now well documented that PSA is also
Because hK2 is a secreted protein, it may be present in
produced by extraprostatic sources (7, 8 ). Several studies
various biological fluids.
have demonstrated the presence of PSA in the periure-
Methods: We analyzed milk samples from lactating
thral and perianal glands (9 –11 ); PSA immunoreactivity
women, amniotic fluid from pregnant women, and
has also been detected in 30 – 60% of female breast tumor
breast cyst fluid from patients with gross breast cystic
cytosolic extracts (12, 13 ) and in breast cancer cell lines
disease, using a highly sensitive and specific immuno-
after stimulation by steroid hormones (14 ). Furthermore,
assay for hK2.
PSA was found in healthy endometrial tissue (15 ), in the
Results: hK2 was present in all three biological fluids.
milk of lactating women (16 ), in breast cyst fluid (17 ), and
We suggest that the female breast may produce hK2 and
in amniotic fluid (18, 19 ).
provide evidence that hK2 may have value as an addi-
The hK2 gene was identified in 1987 by molecular
tional marker for the discrimination between type I and
techniques (20 ). Shortly afterward, it was demonstrated
type II breast cysts.
that the gene is expressed in the prostate (21 ) and the
complete cDNA for hK2 was elucidated (22 ). The de-
duced amino acid sequence revealed a mature hK2 pro-
tein containing 237 amino acids and an approximately
1 Department of Pathology and Laboratory Medicine, Mount Sinai Hospi-
80% sequence identity to PSA. In contrast to PSA, enzyme
tal, Toronto, Ontario, Canada M5G 1X5, and Department of Laboratory
Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
specificity for hK2 is trypsin-like, with selective cleavage
M5G 1L5.
at arginine residues as previously predicted by its amino
2 Department of Biochemistry and Molecular Biology, Faculty of Medicine,
acid sequence (23 ).
University of Oviedo, Oviedo, Spain.
3
The striking homology of hK2 to PSA, combined with
Department of Surgery, Hospital de Jove, Gijon, Spain.
4 Fundacion Tejerina, Madrid, Spain.
*Address correspondence to this author at: Department of Pathology and
Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto,
Ontario,
Canada
M5G
1X5.
Fax
416-586-8628;
e-mail
ediamandis@
5 Nonstandard abbreviations: hK2, human glandular kallikrein; PSA, pros-
mtsinai.on.ca.
tate-specific antigen; BCF, breast cyst fluid; and GCBD, gross cystic breast
Received March 11, 1999; accepted July 7, 1999.
disease.
1774

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Clinical Chemistry 45, No. 10, 1999
1775
the prostate localization of both kallikreins as well as the
with shaking. After the wells were washed, 100 ?L of
androgen regulation of both genes, suggested that they
biotinylated mouse monoclonal detection antibody (code
may have a close physiological relationship. This is
8311; Diagnostic Systems Laboratories) was added to each
strongly supported by recent findings that hK2 cleaves
well and incubated for 1 h. Wells were washed, and
pro-PSA (244 residues) to generate enzymatically active
alkaline phosphatase-labeled streptavidin was added, in-
PSA (237 residues) (24 –26 ). Recombinant hK2, which
cubated for 15 min, and washed again. The alkaline
recently was expressed and purified (27 ), monoclonal
phosphatase activity was measured by adding the sub-
antibodies, and immunological assays developed for hK2
strate diflunisal phosphate, incubating for 10 min, and
with no cross-reactivity to PSA (28 ) allow direct and
then adding a Tb3?-EDTA developing solution. The fluo-
reliable studies on this kallikrein.
rescence was measured on a Cyberfluor 615 Immunoana-
Considering the potential role of hK2 as a physiological
lyzer (MDS Nordion). The hK2 assay has a detection limit
regulator of PSA, we speculated that hK2 may be present,
of 0.006 ?g/L and has ?0.2% cross-reactivity to PSA (32 ).
along with PSA, in tissues and biological fluids of non-
prostatic origin. Very recent studies identifying the ex-
Results
pression of hK2 in the breast carcinoma cell line T-47D
PSA and hK2 in milk
after stimulation by steroid hormones (29 ) and in breast
We analyzed 44 milk samples (1 sample per lactating
tumor extracts and nipple aspirate fluids (30 ) support this
woman), for PSA and 41 samples for hK2 (3 samples were
hypothesis. Therefore, we analyzed milk from lactating
depleted). The time of collection post delivery, the sex of
women, breast cyst fluid (BCF) from women with gross
the newborn, and the age of the mother were available for
cystic breast disease (GCBD), and amniotic fluid with a
all samples; the statistics for the samples are shown in
highly sensitive immunoassay for hK2 that is devoid of
Table 1.
cross-reactivity from PSA.
All but five milk samples contained detectable
amounts of PSA, with a median concentration of 0.084
Materials and Methods
?g/L. Four samples had relatively high concentrations:
samples
11.8, 24, 67, and 111 ?g/L. The distribution of PSA
Breast milk was collected from women post delivery and
concentrations in the milk samples relative to the number
was stored at ?20 °C until analysis. The samples were
of days after delivery, to the sex of the newborn, and to
thawed and centrifuged at 12 000g for 10 min; after
the age of the mother is presented in Fig. 1. Statistical
removal of the top lipid layer, the samples were assayed
analysis indicated that there was a negative correlation
for PSA and hK2. The amniotic fluids analyzed were
[Spearman correlation coefficient (rs) ? ?0.58; P ?0.001]
leftovers from screening programs for fetal abnormalities.
between PSA concentration and postdelivery time, which
They were kept frozen at ?70 °C until analysis. The BCFs
is in accordance with our previous findings (16 ). How-
were obtained by needle aspiration from 104 women with
ever, we found no association between milk PSA and
GCBD. The samples were centrifuged at 12 000g for 10
either sex of newborn (Mann–Whitney nonparametric
min, and the supernatants were stored at ?70 °C until
U-test; Fig. 1B), or maternal age (Spearman correlation;
analysis.
Fig. 1C).
Our study was approved by the Ethics Committee of
hK2 was detected in 35 of the 41 samples analyzed. The
Mount Sinai Hospital, Toronto, Ontario, Canada.
median concentration was 0.021 ?g/L, and the highest
concentrations observed were ?2.2–2.7 ?g/L (in three
immunoassays
samples). In 23 of the samples, the hK2 concentrations
The total PSA concentration in all samples was measured
were 1.5- to 100-fold lower than the corresponding PSA
by an ultrasensitive time-resolved immunofluorometric
concentrations. In seven samples, the hK2 concentration
assay, as described elsewhere (31 ). The PSA assay has a
was higher than PSA (1.5- to 29-fold), whereas in three
detection limit of 0.001 ?g/L and has no detectable
samples, the two kallikreins were present in approxi-
cross-reactivity to hK2, as previously established (32 ). A
mately equal concentrations. Finally, in two samples with
new, time-resolved immunofluorometric assay, recently
undetectable PSA, low concentrations of hK2, close to the
developed in our laboratory, was used to measure hK2
detection limit of our assay, were measured. The distri-
concentrations (32 ). Briefly, the hK2 assay uses a mouse
monoclonal anti-hK2 capture antibody (supplied by Hy-
britech Inc., San Diego, CA, and raised against recombi-
Table 1. Analysis of PSA and hK2 in milk of
nant hK2) immobilized onto polystyrene microtitration
lactating women.
wells at a concentration of 4 mg/L in 100 ?L of coating
Number of
buffer per well (total of 400 ng of capture antibody per
Variable
samples
Mean (SD)
Median
Range
well). After overnight incubation, the plates were washed
PSA, ?g/L
44
5.2 (19.5)
0.084
0–111
and the samples were applied undiluted at a volume of
hK2, ?g/L
41
0.35 (0.66)
0.021
0–2.67
100 ?L, followed immediately by the addition of 50 ?L of
Days post-delivery
44
22 (46)
5
2–243
Mother’s age, years
44
31 (4.1)
31
20–39
assay buffer, and incubated at room temperature for 1 h

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1776
Magklara et al.: hK2 in Biological Fluids
Fig. 1. PSA concentrations in 44 milk samples obtained at various times post delivery (A) and plotted according to the sex of newborn (B) and
maternal age (C).
In A and C, the Spearman correlation coefficient (r ) was used to determine the correlation between the two variables. In B, the horizontal lines indicate the median
s
PSA concentration for each gender; P was determined by Mann–Whitney U-test. Three twin pregnancies were excluded from this analysis.
bution of hK2 concentrations according to the number of
with time after delivery. We found a strong positive
days after delivery, to the sex of the newborn, and to the
correlation between PSA and hK2 concentrations in milk
age of the mother is presented in Fig. 2. We found no
(r ?
s
0.79; P ?0.001; Fig. 3).
statistically significant correlation between hK2 concen-
tration and either the age of the mother or the sex of the
PSA and hK2 in amniotic fluid
newborn, as is the case for PSA. However, hK2 concen-
Amniotic fluid samples (n ? 116) from different gesta-
tration correlates negatively (r ? ?
s
0.48; P ?0.001) with
tional ages (11–25 weeks of gestation as well as terminal)
postdelivery time, suggesting that similar to PSA, hK2
were assayed for PSA and hK2. PSA was detected in all
concentrations in the milk of lactating women decline
samples, with concentrations of 0.001–2 ?g/L; one sample
Fig. 2. hK2 concentrations in 41 milk samples obtained at various times post delivery (A) and plotted according to the sex of newborn (B) and
maternal age (C).
In A and C, the Spearman correlation coefficient (r ) was used to determine the correlation between the two variables. In B, the horizontal lines indicate the median
s
hK2 concentration for each gender; P was determined by Mann–Whitney U-test. Three twin pregnancies were excluded from this analysis.

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Clinical Chemistry 45, No. 10, 1999
1777
Table 2. Analysis of PSA in BCF samples.
PSA, ?g/L
All samples
Type I cysts
Type II cysts
(n ? 103)
(n ? 85)
(n ? 18)
Percentile
0
0.001
0.001
0.001
5
0.003
0.004
0.002
25
0.020
0.021
0.017
50
0.076
0.075
0.076
75
0.564
0.573
0.474
95
14.068
13.361
11.763
100
42.674
42.674
16.705
Mean ? SE
1.70 ? 0.56
1.75 ? 0.65a
1.46 ? 0.94a
a There was no difference in PSA concentration between type I and type II cysts
(P ? 0.65 by Mann–Whitney U-test).
no detectable hK2, no statistical analysis was deemed
necessary.
PSA and hK2 in bcf
According to the K?/Na? ratio, the 103 samples were
Fig. 3. Correlation between PSA and hK2 concentrations in 41 milk
distributed into two groups: type I cysts (with ratio ?1.5)
samples.
and type II cysts (with ratio ?1.5). Eighty-five BCF
Spearman correlation coefficient (r ) was used to determine the correlation
s
between the two variables. The regression equation is: y
samples (82%) were found to belong to type I cysts and 18
? 0.021x ? 0.236,
where y is hK2, and x is PSA.
(18%) to type II cysts.
The PSA concentrations ranged from below the detec-
tion limit of our assay in two samples to 42.7 ?g/L. The
had a concentration of 185 ?g/L. The 10th, 25th, 50th,
frequency distribution of values along with their means
75th, and 90th percentiles for the PSA concentrations are
and medians in all fluids as well as in type I (apocrine)
shown in Fig. 4. There was a positive correlation between
and in type II (flattened) cysts separately are shown in
PSA and gestational age (data not shown), as reported
Table 2. Because the distribution of PSA concentrations
previously (18, 19 ).
was not gaussian, the analysis of differences between the
hK2 was detected in 31 (27%) of the 116 samples
two cyst types was performed with the nonparametric
examined. The highest concentration observed was 0.38
Mann–Whitney U-test. There was no statistically signifi-
?g/L in the sample that had the highest PSA concentra-
cant difference in PSA concentrations between the two
tion (185 ?g/L). Because most of the amniotic fluids had
groups (P ? 0.65).
The hK2 concentration in cyst fluids ranged from
below the detection limit, in eight samples, to 21.2 ?g/L,
with a mean of 1.03 ? 0.26 ?g/L. Using the Mann–
Whitney U-test, we found a statistically significant differ-
Table 3. Analysis of hK2 in BCF samples.
hK2, ?g/L
All samples
Type I cysts
Type II cysts
(n ? 103)
(n ? 85)
(n ? 18)
Percentile
0
0.002
0.003
0.002
5
0.004
0.005
0.003
25
0.019
0.021
0.010
50
0.101
0.136
0.017
75
0.715
0.773
0.237
95
6.120
6.309
2.045
100
21.201
21.201
2.783
Mean ? SE
1.03 ? 0.26
1.19 ? 0.313a
0.27 ? 0.16a
Fig. 4. Distribution of PSA concentrations in amniotic fluid.
a There was a statistically significant difference in hK2 concentration in type I
The box and whiskers display the 10th, 25th, 50th, 75th, and 90th percentiles.
?
vs type II cysts (P ? 0.02 by Mann–Whitney U-test).
, outliers.

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1778
Magklara et al.: hK2 in Biological Fluids
noma cell line T-47D can produce and secrete hK2 after
steroid hormone stimulation (29 ). This cell line was
reported previously to produce substantial amounts of
PSA when stimulated by steroids (14 ). hK2 has also been
detected in breast tumor extracts and in nipple aspirate
fluid, a common breast secretion (30 ). These findings
suggest that the hK2 gene is expressed in breast tissue.
To verify this hypothesis, we first examined milk from
lactating women. The concentrations of circulating ste-
roids are increased during pregnancy, and they could
induce the production of this kallikrein. Yu and Diaman-
dis (16 ) have shown that PSA is present in considerable
concentrations in human milk. We found that the vast
majority of the milk samples examined contained variable
concentrations of hK2, ranging from ?0.006 to 2.67 ?g/L.
Because hK2 is undetectable in serum from healthy fe-
Fig. 5. Relationship between hK2 concentration and cyst type.
males (32 ), we can rule out the possibility of hK2 diffusion
The hK2 concentrations in 103 breast fluids are plotted according to cyst type.
from the circulation into the milk and assume that it is
P was determined by the Mann–Whitney U-test.
secreted by breast epithelial cells. There was no significant
correlation between the hK2 concentration in milk and the
ence between the hK2 values in the two cyst types (P ?
age of the mother or the gender of the newborn, but we
0.02; Table 3 and Fig. 5).
found a declining trend with postdelivery time (Fig. 2) for
No correlation was observed between either PSA or
both PSA and hK2 concentrations (16 ). This is likely
hK2 and the K?/Na? ratio (data not shown). However,
attributable to the decrease of steroid hormone concentra-
there was a significant correlation between PSA and hK2
tions in maternal serum after removal of the placenta. The
concentrations when all samples were considered (r ?
physiological role of hK2 in human breast milk currently
s
0.37; P ?0.001) and when type I cysts were considered
is unknown. hK2 may serve as regulator of PSA activity,
separately (r ?
for which an involvement in the growth of healthy breast
s
0.39; P ?0.001). There was no significant
correlation between PSA and hK2 concentrations in type
tissue has been proposed (16 ), or it may have a physio-
II cysts (r ?
logical activity of its own because it has been shown to
s
0.20; P ? 0.43; Fig. 6).
cleave insulin-like growth factor-binding proteins even
Discussion
more rapidly than PSA (33 ).
hK2 is a serine protease closely related to PSA. hK2 is
The presence of hK2 in amniotic fluid was verified in
primarily, if not exclusively, expressed in the prostate
only a small proportion of samples, and the concentra-
gland. It recently has been reported that the breast carci-
tions were relatively low. This is a notable difference
Fig. 6. Correlation between PSA and hK2 concentrations in all BCFs (A), in type I cysts (B), and in type II cysts (C).
Spearman correlation coefficient (r ) was used to determine the correlation between the two variables.
s

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Clinical Chemistry 45, No. 10, 1999
1779
between PSA and hK2, which seem to coexist in all other
identified insulin-like growth factor-I in BCF. hK2 has
breast secretions. The source of hK2, as well as that of
been shown to cleave insulin-like growth factor-binding
PSA, in amniotic fluid remains unclear.
proteins, which suggests a role similar to that proposed
In this study, we report for the first time that women
for PSA (33 ). The significant correlation between hK2 and
with GCBD produce and accumulate in the BCF relatively
PSA in all the samples tested suggests that the factor(s)
large amounts of hK2. This finding, in conjunction with
that control their production and/or the mechanism by
our findings of hK2 present in nipple aspirate fluid and
which they enter the BCF are similar.
milk, further suggests that the breast epithelium secretes
hK2, an enzyme that was originally defined as prostate
In summary, considering the fact that the inactive precur-
specific. Although breast cysts by themselves are not
sor of PSA, pro-PSA, is rapidly converted to active PSA by
considered precancerous lesions, they have been associ-
hK2, suggesting an important in vivo regulatory function
ated with a higher risk for developing breast cancer
by hK2 on PSA activity, we hypothesized that hK2 may
(34, 35 ). Taking into account that benign and malignant
exist in biological fluids in which PSA has already been
breast diseases could have some common pathogenetic
detected, such as milk, BCF, and amniotic fluid. We report
factors, the analysis of BCF, in which the metabolic
here the consistent presence of hK2 in breast milk and
products of the cells lining the cysts accumulate, could
BCF at concentrations that strongly support production
provide additional information about the environment of
from the breast tissue, possibly after steroid stimulation.
the breast tissue, regarding focal lesions prone to malig-
This finding is in accordance with the notion that PSA and
nant transformation. Examination of human BCF reveals
hK2 coexist in biological tissues, correlating strongly and
two major subgroups of cysts: type I, with a high K?/Na?
possibly acting synergistically. The PSA and hK2 concen-
ratio and large amounts of dehydroepiandrosterone sul-
trations in breast secretions are not always proportionate,
fate, lined with apocrine epithelium and resembling the
as is the case for seminal plasma (32 ). However, the
intracellular microenvironment; and type II, with a low
PSA/hK2 ratio in milk was 1.5–100, with a median of 2,
K?/Na? ratio and lower amounts of dehydroepiandros-
whereas in BCF the median was 1; in seminal plasma, hK2
terone sulfate, lined with flattened epithelium. A third
is present at concentrations 100- to 500-fold lower than
population with intermediate K? and Na? concentrations
PSA (32 ). It appears that both kallikreins are compart-
has also been reported (36 ).
mentalized in breast and prostate tissue and secretions,
In the present study, no significant difference was
but their concentrations in women do not differ as greatly
found in the mean values of PSA concentrations between
as in men. More studies are needed to evaluate whether
the two cyst subgroups, which is in accordance with the
hK2 has some prognostic/diagnostic value in benign and
data of Lai et al. (37 ) and Filella et al. (38 ); nevertheless,
malignant breast disease. Clearly, the concurrent expres-
we and others have reported that there is a small differ-
sion of PSA and hK2 in the female breast is of high interest
ence in the mean PSA concentrations in the two types of
and their physiological role in this tissue needs further
cysts (39, 40 ). On the other hand, hK2 not only shows a
investigation.
wide range of concentrations in BCF (Table 3) but a
differential distribution of values as well, which is consis-
tent with the presence of distinct subpopulations of cysts.
We thank Cathy Buller for collecting milk samples and
The fact that hK2 is detected at higher concentrations in
Herb Yu for assistance in the statistical analysis. We are
type I cysts (Fig. 5) suggests that the epithelium lining this
grateful to Dr. R. Wolfert from Hybritech Inc., San Diego,
group of cysts may be especially active in hK2 production
CA, for providing one hK2 antibody.
because of its high content of steroid hormones, mainly
androgens. This observation is interesting because it
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