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Immunohistochemical Evidence of Loss of PTEN
Expression in Primary Ductal Adenocarcinomas of
the Breast
Aurel Perren,*
†‡
Liang-Ping Weng,*
Alexander H. Boag,
§
Ulricke Ziebold,
i
Kosha Thakore,*
†
Patricia L. M. Dahia,*
†
Paul Komminoth,
‡
Jacqueline A. Lees,
i
Lois M. Mulligan,
§¶
George L. Mutter,** and
Charis Eng*
††
From the Clinical Cancer Genetics and Human Cancer Genetics
Programs,*Ohio State University Comprehensive Cancer Center,
Columbus, Ohio; the Dana-Farber Cancer Institute and Harvard
Medical School,
†
Boston, Massachusetts; the Department of
Pathology,
‡
University of Zu¨ rich, Zu¨ rich, Switzerland; the
Departments of Pathology
§
and Paediatrics,
¶
Queen’s University,
Kingston, Ontario, Canada; the Department of Biology,
i
Cancer
Research Center, Massachusetts Institute of Technology,
Cambridge, Massachusetts; the Department of Pathology,**
Brigham and Women’s Hospital and Harvard Medical School,
Boston, Massachusetts; and the CRC Human Cancer Genetics
Research Group,
††
University of Cambridge, Cambridge,
United Kingdom
Germline mutations in PTEN, encoding a dual-speci-
ficity phosphatase on 10q23.3, cause Cowden syn-
drome (CS), which is characterized by a high risk of
breast and thyroid cancers. Loss of heterozygosity of
10q22–24 markers and somatic PTEN mutations have
been found to a greater or lesser extent in a variety of
sporadic component and noncomponent cancers of
CS. Among several series of sporadic breast carcino-
mas, the frequency of loss of flanking markers
around PTEN is approximately 30 to 40%, and the
somatic intragenic PTEN mutation frequency is <5%.
In this study, we analyzed PTEN expression in 33
sporadic primary breast carcinoma samples using im-
munohistochemistry and correlated this to structural
studies at the molecular level. Normal mammary tis-
sue had a distinctive pattern of expression: myoepi-
thelial cells uniformly showed strong PTEN expres-
sion. The PTEN protein level in mammary epithelial
cells was variable. Ductal hyperplasia with and with-
out atypia exhibited higher PTEN protein levels than
normal mammary epithelial cells. Among the 33 car-
cinoma samples, 5 (15%) were immunohistochemi-
cally PTEN-negative; 6 (18%) had reduced staining,
and the rest were PTEN-positive. In the PTEN-positive
tumors as well as in normal epithelium, the protein
was localized in the cytoplasm and in the nucleus (or
nuclear membrane). Among the immunostain nega-
tive group, all had hemizygous PTEN deletion but no
structural alteration of the remaining allele. Thus, in
these cases, an epigenetic phenomenon such as hy-
permethylation, decreased protein synthesis or in-
creased protein degradation may be involved. In the
cases with reduced staining, 5 of 6 had hemizygous
PTEN deletion and 1 did not have any structural ab-
normality. Finally, clinicopathological features were
analyzed against PTEN protein expression. Three of
the 5 PTEN immunostain-negative carcinomas were
also both estrogen and progesterone receptor-nega-
tive, whereas only 5 of 22 of the PTEN-positive group
were double receptor-negative. The significance of
this last observation requires further study. (Am J
Pathol 1999, 155:1253–1260)
The tumor suppressor gene PTEN, encoding a dual-
specificity phosphatase, has been cloned and mapped
to chromosome 10q23.3.
1–3
Germline PTEN mutations
are found in the autosomal dominant Cowden syndrome
(CS), which is characterized by multiple hamartomas in-
volving many organ systems as well as an increased risk
of developing breast and thyroid cancers.
4,5
Loss of het-
erozygosity (LOH) of markers at 10q23–25 is a frequent
event (30 –50%) in endometrial cancer,
6–9
glioblas-
toma,
10
and breast cancer.
11–13
Somatic intragenic mu-
tations of PTEN are a frequent event in endometrial car-
cinomas,
6–9
malignant gliomas,
14–17
and melanomas.
18
However, unlike endometrial carcinoma and glioblas-
toma, only a very small fraction (,5%) of the 40% of
primary breast cancers showing allelic loss in this region
also have mutations in the remaining allele,
11–13,19
de-
Supported in part by grants from the American Cancer Society (RPG-97-
064-01-VM and RPG98-211-01-CCE), the Department of Defense Breast
Cancer Research Program (DAMD17-98-1-8058), the Concert for the
Cure (to C. E.), the National Cancer Institute (P30CA16058, Comprehen-
sive Cancer Center), and the Canadian Breast Cancer Research Initiative
(to L. M. M.). P. L. M. D. is a Postdoctoral Fellow of the Susan G. Komen
Breast Cancer Research Foundation (to C. E.) and A. P. is a Fellow of the
Lydia Hochstrasser-Stiftung, Zu¨ rich, Switzerland (to P. K.).
Accepted for publication June 15, 1999.
Address reprint requests to Charis Eng, Human Cancer Genetics Pro-
gram, Ohio State University Comprehensive Cancer Center, 690C Medi-
cal Research Facility, 420 W. 12th Avenue, Columbus, Ohio 43210. E-
mail: eng-1@medctr.osu.edu.
American Journal of Pathology, Vol. 155, No. 4, October 1999
Copyright © American Society for Investigative Pathology
1253
spite the fact that females with CS have a #50% lifetime
risk of developing breast cancer.
5,20,21
In contrast to
these analyses based on primary breast carcinomas,
initial studies using breast cancer cell lines seemed to
show that a large proportion have biallelic loss of
PTEN.
1,3
Investigators, therefore, questioned whether
loss of one PTEN allele (haploinsufficiency) is sufficient
for tumorigenesis or whether inactivation of the second
allele might occur through epigenetic rather than muta-
tional events.
We report a study of PTEN expression using immuno-
histochemical methods in a series of 33 primary human
breast tumors. This is a powerful method because it
provides an internal control comparing the staining of
tumor tissue to that of the adjacent normal breast tissue.
We also began to explore the association of PTEN ex-
pression with genomic PTEN status and clinicopatholog-
ical features.
Materials and Methods
Breast Carcinoma Samples
Paraffin blocks of 33 unselected sporadic primary ductal
breast carcinomas were drawn from the files of the King-
ston General Hospital (Kingston, ON, Canada). LOH
analysis with seven microsatellite markers known to map
to the 10q23 interval and flanking PTEN as well as PTEN
mutation analysis have been performed previously.
13
Of
the 33 women diagnosed with primary mammary adeno-
carcinomas, 4 were diagnosed before the age of 50. The
tumors ranged in size from 1 to 6 cm. There were 2 well
differentiated, 13 moderately differentiated, and 18
poorly differentiated tumors. Seven of the 13 women had
regional lymph node involvement at presentation.
Immunohistochemistry
The monoclonal antibody 6H2.1 raised against the last
100 C-terminal amino acids of PTEN (Ziebold and Lees,
unpublished) was used in all immunohistochemical
analyses.
The tissue samples were fixed by immersion in 10%
buffered formalin and embedded in paraffin according to
standard procedures.
22
Four-millimeter sections were cut
and mounted on Superfrost Plus slides (Fisher Scientific,
Pittsburgh, PA). Immunostaining was performed essen-
tially as described.
22–24
In summary, the sections were
deparaffinized and hydrated by passing through xylene
and a graded series of ethanol. Antigen retrieval was
performed for 20 minutes at 98°C in 0.01 mol/L sodium
citrate buffer, pH 6.4, in a microwave oven. Incubating
the sections in 0.3% hydrogen peroxide for 30 minutes
blocked endogenous peroxidase activity. After blocking
for 30 minutes in 0.75% normal horse serum the sections
were incubated with 6H2.1 (dilution 1:100) for 1 hour at
room temperature. The sections were washed in Tris-
buffered saline and then incubated with biotinylated
horse anti-mouse IgG followed by avidin peroxidase us-
ing the Vectastain ABC elite kit (Vector Laboratories,
Burlingame, CA). The chromogenic reaction was carried
out with 3–39diaminobenzidine using nickel cobalt am-
plification,
25
which gives a black product. After counter-
staining with Nuclear Fast Red (Rowley Biochemical,
Danvers, MA) and mounting, the slides were evaluated
under a light microscope. According to the amount of
staining, the tumors were divided in three groups: the
group assigned 11 showed increased or equal staining
intensity compared to the corresponding normal tissue;
the group assigned 1had decreased staining intensity;
and the group assigned 2had no trace of staining.
A series of commercially available cell lines with known
PTEN status was used as positive and negative controls
to prove antibody specificity by immunohistochemistry:
Balb C/3T3, Nalm6, DU145, MDA-MB-468, A172, and
PC3 (see Results). In addition, the U2OS osteosarcoma
cell line was transfected with full length PTEN cDNA
expression construct as a further positive control.
Incubating the sections in the absence of antibody as
well as preincubation during 2 hours at 37°C of the anti-
body with recombinant PTEN protein led to negative re-
sults (data not shown).
Western Blot Analysis
As biochemical proof of antibody specificity for PTEN,
total protein lysates were obtained
26
(Dahia, 1999 #955)
from a series of commercially available cell lines (Amer-
ican Type Culture Collection, Manassas, VA), for which
PTEN status is known: MCF-7, T47D, MDA-MB-435S,
ZR-75-1, BT-549, and MDA-MB-468 (see Results). In ad-
dition, as an additional positive control, the wild-type full
length human PTEN cDNA sequence was cloned into the
mammalian expression vector pUHD10-3, which con-
tains a tetracycline-suppressible promoter (Gossen,
1992 #1065), and stably transfected into the MCF7/T-off
(Clontech, Palo Alto, CA) breast cancer cell line. After 24
hours of tetracycline withdrawal for purposes of PTEN
induction, protein lysates were collected for Western blot
analysis as well. Western blot analysis was performed as
previously described,
26
except that 6H2.1 was used at a
1:250 dilution. Control antibody was against
a
-tubulin
and used at 1:1000 dilution.
LOH Analysis
All breast carcinoma samples have been previously eval-
uated for LOH with markers closely flanking, but not
within, PTEN.
13
In the event that our immunohistochemi-
cal results seemed to be discordant with the molecular
analyses, further LOH analysis was performed using
markers within PTEN itself as previously described.
26,27
Potential hemizygosity at the PTEN locus was assessed
by screening for a T/G polymorphism within PTEN intron
8 detected by differential digestion with the restriction
endonuclease HincII as previously described
27
and
the intragenic markers D10S2491, AFM086wg9, and
D10S2492.
1254 Perren et al
AJP October 1999, Vol. 155, No. 4
Results
Specificity of Monoclonal Antibody 6H2.1
Because this study relied on a monoclonal antibody,
6H2.1, specific recognition of PTEN by this antibody is
crucial. Western blot analysis using a series of breast
cancer lines with known PTEN status and the 6H2.1 anti-
PTEN monoclonal antibody demonstrated the specificity
of this antibody (Figure 1). Western analysis of three
PTEN1/1lines, MCF-7, T-47D, and MDA-MB-435S, re-
vealed a single band at the molecular weight predicted
for PTEN. After induction of MCF-7/PTEN, increased ex-
pression of PTEN was evidenced by an increased band
intensity (Figure 1). In contrast, ZR-75-1, with a hemizy-
gous deletion of PTEN and a missense mutation in the
remaining allele, yielded a weak band of the expected
size. BT-549 and MDA-MB-468, which are null for PTEN,
had no signal. No nonspecific bands were noted. Control
blot with anti-
a
-tubulin antibody revealed signals for all
lines.
To test the suitability of the antibody for immunohisto-
chemistry, we used PTEN-transfected U2OS cells as well
as a series of cell lines expressing PTEN (Balb C/3T3,
Nalm6, DU145) as positive controls. MDA-MB-468, a breast
cancer cell line with a hemizygous deletion of PTEN and a
truncating mutation of the remaining allele, A172, a glioblas-
toma cell line with loss of one PTEN allele and a truncating
mutation in exon 2 of the remaining allele and PC3, a
prostate cell line with homozygous deletion of PTEN,
were used as negative controls (data not shown).
PTEN Immunohistochemistry in Primary
Breast Carcinomas
Samples from 33 sporadic primary breast carcinomas,
which had been examined previously for LOH of markers
flanking PTEN as well as somatic PTEN mutations,
13
were
subjected to immunohistochemical analysis using a
monoclonal antibody, 6H2.1, raised against the terminal
100 amino acids of human PTEN. Of the 33 total cancers,
29 had accompanying normal tissue; in each of the 29
samples, the normal glandular epithelium showed immu-
noreactivity to 6H2.1. Interestingly, there was a distinctive
staining pattern in the normal tissue. The myoepithelial
cells of the normal ducts showed the strongest signal with
a nuclear predominance (Figure 2B). In contrast, the
amount of staining in the epithelial cell layer was variable.
Areas of epithelial ductal hyperplasia with and without
atypia stained more strongly than the normal epithelia
(Figure 2A). Endothelial cells, especially within neovas-
cular capillaries, and nerves showed strong PTEN ex-
pression and were useful as internal positive controls.
Of 33 breast carcinoma samples, 5 (15%) lost all PTEN
immunoreactivity and showed negative immunostaining,
graded 2(Table 1 and Figure 2, E and F). In each of
these 5 cases, adjacent non-neoplastic glands (Figure
2F) as well as enclosed non-neoplastic ducts (Figure 2 E)
stained positively. Interestingly, the cells within the des-
moplastic reaction surrounding each of these 5 carcino-
mas had high PTEN expression. Six of the 33 (18%)
breast cancer specimens stained weakly, graded 1,in
Figure 1. Western blot of 7 breast cancer cell lines using the anti-PTEN monoclonal antibody 6H2.1 (left panel) and using the anti-
a
-tubulin antibody as a control
(right panel). MCF-7, T-47D, and MDA-MB-435S have endogenous PTEN. BT-549 and MDA-MB-468 are PTEN-null. ZR-75-1 has monoallelic PTEN deletion and
a missense mutation on the remaining allele. MCF-7/PTEN is the MCF-7 line transfected with a wild-type PTEN construct and a tetracycline-inducible promoter
after withdrawal of tetracycline and, hence, induced expression of PTEN.
PTEN Immunohistochemistry in Breast Carcinoma 1255
AJP October 1999, Vol. 155, No. 4
comparison to the normal tissue (Table 1 and Figure 3).
One of these tumors (Sample 40, Table 1) showed pos-
itive immunostaining in the intraductal component,
whereas the adjacent invasive component lost almost all
PTEN protein expression (Figure 3A). The remaining 22
(66%) tumors stained positively, graded 11 (increased
staining compared to normal glands). All these tumors
showed homogeneous PTEN immunoreactivity through-
out the examined section. PTEN immunoreactivity in
these 22 tumors as well as their corresponding normal
and hyperplastic breast tissue involved the cytoplasmic
and nuclear (most likely nuclear membrane) compart-
ment of the cells.
Comparison of Immunohistochemical and
Structural Mutation Data
Immunohistochemical evidence of PTEN expression was
absent or weak in a total of 11 (33%) of 33 breast carci-
nomas. These breast carcinomas had been previously
examined for LOH of markers flanking PTEN and also for
intragenic PTEN mutations;
13
40% demonstrated LOH
but there were no intragenic PTEN mutations or biallelic
deletion. Whether there is a one-to-one concordance be-
tween molecular and immunohistochemical observations
is further explored in this report.
LOH analysis for markers in the 10q22–24 interval was
previously performed using seven microsatellite markers
(centromeric to telomeric): D10S579, D10S215,
D10S1765, D10S541, D10S1735, D10S1739, and
D10S564.
13
PTEN lies between D10S1765 and D10S541,
a genetic distance of 1 cM but a physical distance of only
several hundred kilobasepairs. For purposes of this
study, to compare the immunohistochemical data to the
LOH data, PTEN was considered to be physically deleted
only when one or more immediately flanking (informative)
markers centromeric and telomeric of PTEN showed
LOH. Using this strict and conservative interpretation for
monoallelic PTEN deletion, 6 of the tumors were shown to
have a loss of one allele of the PTEN gene, another 7 were
shown to have a loss flanking one side of (which may or
Figure 2. A: Ductal hyperplasia ( case 58) with increased staining in the epithelial layer (original magnification, 360). B: Normal breast glands (case 46) with
predominantly nuclear staining in the myoepithelial layer (original magnification, 360). C(case 48) and D(case 43): Ductal carcinoma with strong PTEN staining
(11, original magnification, 330). E: Ductal PTEN-negative carcinoma (arrowhead, case 58) and surrounding normal duct (arrow). Original magnification, 330.
F: Ductal PTEN-negative carcinoma (arrowhead, case 46) with non-neoplastic normal duct (arrow). Original magnification, 330.
Table 1. Correlation between PTEN Immunostaining and
PTEN and/or 10q22-23 LOH
PTEN
Immuno
11
PTEN
Immuno
1
PTEN
Immuno
2
LOH 59Markers 4* 2 4
LOH 39Markers 1* 2 5
ROH Flank Markers 18 2* 0
Total Tumors 22 6 5
Correlation between PTEN immunostaining and LOH of 59and/or 39
flanking markers.
Concordance 82%.
LOH, loss of heterozygosity; ROH, retention of heterozygosity.
*Apparent discordance 18%.
1256 Perren et al
AJP October 1999, Vol. 155, No. 4
may not include) PTEN. For these latter 7 tumors, poten-
tial hemizygosity at the PTEN locus was further assessed
by screening for a T/G polymorphism within PTEN intron
8 (IVS8132T/G), detected by differential digestion with
the restriction endonuclease HincII, and the intragenic
polymorphic markers AFM086wg9, D10S2491, and
D10S2492. AFM086wg9 lies in intron 2 of PTEN. The likely
intragenic marker order is centromere 2D10S2491 2
AFM086wg9 2D10S2492/IVS8132T/G 2telomere
(Marsh and Eng, unpublished).
Of the 5 breast carcinomas that exhibited no immuno-
histochemical evidence of PTEN expression (graded 2),
4 showed extensive LOH of markers flanking PTEN and
hence, PTEN itself (Table 1, Column 3 and Table 3). The
fifth carcinoma had LOH on the telomeric side (D10S541)
of PTEN. Further molecular analysis revealed retention of
heterozygosity at AFM086wg9 but LOH at the IVS8132T/G
polymorphism, suggesting hemizygous deletion of the 39
end of PTEN. Therefore, all 5 breast carcinomas that had
negative PTEN immunostaining also had hemizygous
PTEN deletion (Table 3). None of these 5 had biallelic
deletion of PTEN nor did they have a second intragenic
PTEN hit, ie, mutation of the remaining allele.
Of the 6 carcinomas that had weak PTEN immunostain-
ing, graded 1, 4 had been previously shown to have LOH
of markers flanking one side or the other of PTEN and 2
Figure 3. Cases with weak staining (arrows in Cand F, non-neoplastic duct; arrow in E, blood vessel). A: Ductal carcinoma (case 40) showing no staining
(graded 2) in the invasive component (top) adjacent to immunostain-positive intraductal component (bottom). B: Case 66. C: Case 59. D: Case 57. E: Case 45.
Original magnification, 330.
PTEN Immunohistochemistry in Breast Carcinoma 1257
AJP October 1999, Vol. 155, No. 4
showed no LOH of flanking markers (Tables 2 and 3).
Further LOH analysis within PTEN revealed that the 4
carcinomas with LOH of markers flanking one side of the
gene also had LOH of at least one of the intragenic
markers (Table 2). Thus, these 4 tumors with decreased
immunostaining seemed to have hemizygous deletion of
PTEN or at least part of it. In the remaining two carcino-
mas without LOH of markers immediately flanking the
gene, further analyses within the gene were uninformative
or showed retention of heterozygosity (Tumors 45 and 57,
Tables 2 and 3). In all likelihood, PTEN might not be
altered at the structural level in that particular tumor.
Among the remaining 22 carcinomas that showed im-
munohistochemical evidence of strong PTEN expression
(increased staining compared to normal mammary
glands), 18 (82%) showed no LOH and biallelic presence
of PTEN was demonstrated (Table 1). There were 4 tu-
mors that seemed to be immunostained (grade 11), yet
showed LOH flanking PTEN (Tables 2 and 3). However, it
should be noted that 3 of these 4 tumors had LOH of
D10S1765 immediately centromeric of PTEN but with ei-
ther retention of heterozygosity or noninformativeness at
D10S541 immediately 39of the gene. Further LOH anal-
ysis within PTEN corroborates the previous observations
(Table 2): in tumor 6, 39markers within the gene showed
retention of heterozygosity and a 59marker (AFM086wg9)
showed LOH; in tumor 5, where D10S1765 showed LOH,
markers within the gene (AFM086wg9 and D10S2492)
and 39of the gene (D10S541) all showed retention of
heterozygosity. Similarly, tumor 9, which had LOH at
D10S1765, had 3 of 4 intragenic markers with retention of
heterozygosity. Tumor 53 was unusual in that both
D10S1765 and D10S541 had LOH, although molecular
analysis demonstrated all 4 intragenic markers with re-
tained heterozygosity.
Correlation of PTEN Immunohistochemistry and
Clinicopathological Parameters
PTEN immunostaining status was compared with such
clinicopathological parameters as age at diagnosis, size
of primary tumor, tumor grade, lymph node status, and
estrogen receptor and progesterone receptor status. Be-
cause of the relatively small numbers, especially in the
context of subset analyses, no conclusions could be
drawn with confidence from our observed correlations.
The most interesting association seemed to be that be-
tween PTEN expression and hormone receptor status
(Table 4). Three of the 5 carcinomas (67%) that had no
PTEN protein were estrogen and progesterone receptor-
negative compared to 5 of 22 (23%; P,0.05 Fisher’s
exact test) in the PTEN-immunopositive samples. All 6
carcinomas that had weak PTEN staining were estrogen
and progesterone receptor-positive. Other trends are
also noteworthy. Although there were only 2 grade I tu-
mors, both had high PTEN expression. All 5 tumors that
were 1.5 cm or smaller had high levels of PTEN protein.
Table 2. Analysis of Correlation between PTEN Immunostaining and PTEN Intragenic LOH in Cases with Decreased
Immunostaining and Apparently Discordant Tumors
Tumor
Immuno-
staining score
PTEN
10q22-23 Markers
S1765 S2491 AFM086 S2492 IVS8 S541
41 11 LOH NI LOH ROH ROH NI
52 11 LOH NI ROH ROH N/A ROH
53 11 LOH ROH ROH ROH ROH LOH
50 11 LOH ROH ROH NI ROH NI
40 1LOH N/A NI N/A LOH ROH
59 1LOH N/A LOH N/A LOH ROH
66 1ROH NI LOH N/A N/A LOH
57 1ROH LOH LOH N/A N/A ROH
55 1ROH NI NI N/A LOH LOH
45 1ROH NI ROH NI ROH ROH
Tumor numbers correspond to those of Feilotter et al.
13
LOH, loss of heterozygosity; ROH, retention of heterozygosity; NI, not informative (germline homozygosity at marker); N/A, not applicable or not
done.
Table 3. Summary of PTEN Expression by
Immunohistochemistry Compared to Molecular
Analysis
PTEN Expression LOH* ROH
PTEN250
PTEN151
PTEN11 121
*LOH of both flanking markers or a minimum of LOH of one
intragenic marker.
Table 4. Estrogen/Progesterone Receptor Status of Breast
Carcinomas by PTEN Immunostaining Status
PTEN IHC status ER/PR 2ER/PR 1
Negative (2)3 2
Decreased (1)0 7
Positive (11)5 16
ER, estrogen receptor; PR, progesterone receptor.
An equivocal positive receptor status (n55) was scored as a
positive.
1258 Perren et al
AJP October 1999, Vol. 155, No. 4
Discussion
In this first report of immunohistochemical analyses of
PTEN expression in sporadic primary breast carcinomas,
we found that 33% of these tumors had either no or
decreased expression of PTEN, which generally ap-
peared to correlate with structural monoallelic deletion of
the gene. Although it is understandable that tumors with
monoallelic loss of PTEN have decreased PTEN expres-
sion at the protein level, one must explain the 5 samples
with no immunoreactivity and structural PTEN hemizygos-
ity. None of these samples was found to have intragenic
PTEN mutations in the remaining allele, either. It is more
than plausible, therefore, that an epigenetic phenome-
non, such as hypermethylation of the promoter region
28
and decreased protein synthesis or increased protein
turnover,
26
might be inactivating the remaining allele.
Similarly, for the tumor (case 45) with decreased staining
but no structural PTEN deletion, similar hypotheses may
be raised. Other explanations include point mutations in
the putative promoter of the remaining allele or normal
tissue contamination of the breast samples, thus giving
pseudo-hemizygosity in the face of real homozygous de-
letion. The latter can be discarded because very careful
microdissection of the carcinoma components was per-
formed by a pathologist with extensive experience in
microdissection. Further, since the pattern of all positive
and negative tumors was homogeneous, regional PTEN
deletions in tumor subclones are very unlikely. Con-
versely, the observation of rare immunopositive tumors
(n54) which appear to have LOH of flanking markers
can be plausibly explained as well: at least in 3 informa-
tive tumors, no deletion of the gene proper or no deletion
of most of the 39end of the gene has occurred. Hence,
the monoclonal antibody, which is raised against the C
terminus of PTEN, would still immunostain these samples
positively. In this situation, therefore, incomplete 59dele-
tion of PTEN might still be associated with translation of a
truncated immunocompetent PTEN protein. In summary,
while structural deletion or mutation of PTEN can lead to
decreased PTEN protein levels, other mechanisms which
lead to complete loss of PTEN expression seem to be
prominent as well, at least in the breast carcinoma model.
Whether loss of PTEN expression is an early or late
event in breast carcinogenesis is still controversial, al-
though preliminary reports suggest that it is a late
event.
11
The observation that loss of PTEN expression is
correlated with a negative estrogen and progesterone
status and that both grade I tumors had strong PTEN
expression also strengthen this hypothesis. There is no
doubt that these latter clinicopathological observations
need to be investigated further. Nonetheless, these data
in toto argue that despite the observation that germline
PTEN mutations cause Cowden syndrome,
4
somatic
PTEN mutation or functional loss of PTEN expression is
associated with tumor progression and not tumor initia-
tion, at least in the breast cancer model. It is also clear
from our and other data that breast carcinogenesis does
not rely uniformly on the involvement of the PTEN path-
way, although how PTEN plays a role in various aspects
of normal development and in the pathogenesis of breast
carcinoma is not straightforward.
Acknowledgments
We thank Jeff FitzGerald for technical assistance, Dr.
Oliver Gimm for expert administrative assistance and
many members of CE’s laboratory for critical review of the
manuscript.
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