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Original Studies |
Westmead Institute for Cancer Research, University of Sydney, Westmead Hospital, Westmead, New South Wales 2145, Australia
Address all correspondence and requests for reprints to: Dr. Rosemary Balleine, Westmead Institute for Cancer Research, University of Sydney, Westmead Hospital, Westmead, New South Wales 2145, Australia. E-mail: rosemary{at}hemonc.wh.su.edu.au
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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2ER and 7ER,
94% containing the 4ER, and 83% containing the
5ER. In 25 of these cases, which were also PR positive,
the most highly expressed PR variants, the 4PR,
6PR, and 4/2PR, a transcript from which a
126-bp portion of PR exon 4 was deleted, were detected in over 90% of
the cases. The alternatively spliced ER variants were expressed at
higher relative levels than the PR species, which had mean levels of
expression less than 10% that of wild-type PR. The most abundant
species was the 7ER, which was present at levels ranging
from 29–83% of the wild type. There was no relationship between the
level of 7ER in individual tumors and the pattern of
expression of the estrogen-responsive proteins PR and pS2. The common
profile of alternatively spliced ER and PR transcripts in breast tumors
means that this feature cannot be used as a discriminator of hormone
responsiveness or other clinical end points. Further, the low level of
expression of the majority of variant species calls into question their
potential for impacting significantly on receptor function. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The discrepancy between the presence of receptors and the likelihood of
a response to endocrine agents in breast cancer has led to speculation
that breast tumors may contain forms of ER or PR with aberrant
function. To investigate this possibility, the ER has been extensively
examined at the messenger ribonucleic acid (RNA) level, and it has
emerged that the primary ER transcript in both normal and malignant
tissue is commonly subjected to a number of alternative splicing events
that give rise to a range of truncated transcripts (7). Since their
discovery, there has been speculation that these transcripts may encode
ER variants that are clinically important in breast cancer, and
functional correlates of some of these have been examined in
vitro. One species in particular, lacking exon 5 of the ER
transcript, (5ER), has generated considerable interest,
as it has been found to have low level constitutive transcriptional
activity (8), and relative to wild-type ER function, both dominant
positive (9) and dominant negative (10) activities have been ascribed
to the 5ER in specific experimental
circumstances. Dominant negative activity has also been attributed to
the exon 7-deleted (7ER) (11), exon 3-deleted
(3ER) (12, 13), and exon 4-deleted
(4ER) (14) ER variants in some expression
systems. Analogous to ER, the existence of alternatively spliced
transcripts of PR has been demonstrated recently in breast tumors (15, 16), and in vitro functional studies have provided evidence
that PR variants lacking exon 6 (6PR) and
exons 5 and 6 (5+6) of the PR sequence may have
dominant negative activity relative to that of the wild-type receptor
(17).
Despite intriguing functional data from in vitro
experiments, attempts to correlate the expression of alternatively
spliced ER transcripts with clinical features of breast tumors have not
clarified the role variant receptors may play in hormone resistance or
disease progression. In a study by Zhang et al. the presence
of multiple, alternatively spliced ER transcripts was documented in a
series of 109 primary breast tumors, and no correlation between the
presence of these variants and clinicopathological variables or disease
recurrence was found (18). In two small studies, modest increases in
the level of the 5ER variant have been
reported in breast tumors compared with that in normal breast (19) and
in recurrent breast cancer compared with that in primary tumors (20).
The level of this variant was not elevated in tamoxifen-resistant
tumors, however (21), nor was it related to disease-free survival in
patients treated with adjuvant tamoxifen (18). Erenburg et
al. have reported that breast tumors express significantly lower
levels of the 3ER variant transcript than normal breast
specimens and have proposed on the basis of the dominant negative
activity of this receptor in vitro that this difference may
play a role in the malignant phenotype (13).
To begin to understand this issue, the broader significance of splicing of receptor transcripts needs to be considered. The question of whether alternative splicing is a general phenomenon in breast tumors and whether tumors that contain alternatively spliced ER transcripts also contain alternatively spliced PR transcripts in particular needs to be addressed. The aim of this study was to document alternatively spliced ER and PR transcripts in a series of breast tumors and to determine whether the coexpression of alternatively spliced ER and PR transcripts was related to receptor function and, hence, hormone sensitivity in breast cancer.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Tumor samples were taken from 35 women with primary carcinoma of the breast treated at Westmead Hospital (Sydney, Australia). The patients ranged in age from 42–83 yr (mean, 60.8).
Detection of ER and PR
ER and PR measurements were made as part of the routine pathological assessment of tumors by enzyme immunoassay (EIA) using the Abbott ER EIA and PgR EIA kits (Abbott Laboratories, Diagnostics Division, North Chicago, IL), as previously described (22). ER or PR values of 15 fmol/mg protein or greater by EIA were deemed positive. The precise quantity of receptors in samples whose ER or PR content was higher than the upper limit of the standard curve was not determined; instead, these were reported as greater than the highest measurable value only. For the purposes of this analysis, this highest value has been used to approximate ER and PR content in such cases. The ER status of tumors was confirmed by immunohistochemical staining of paraffin-embedded tumor sections using a mouse monoclonal anti-human ER antibody Dako-ER 1D5 (Dako Corp., Carpinteria, CA) after microwave antigen retrieval.
Detection of pS2
The estrogen-responsive protein pS2 was detected by immunohistochemistry in paraffin-embedded tumor sections. Sections were incubated for 10 min at 37 C in a 0.05% (wt/vol) solution of protease, (Sigma Pronase Protease Type E, Sigma-Aldrich Co., Castle Hill, Australia) before incubation with a polyclonal anti-pS2 antibody, NCL-pS2 (Novocastra Laboratories Ltd., Newcastle-Upon-Tyne, UK), overnight. After blocking with 3% hydrogen peroxide, a secondary biotin-conjugated antirabbit Ig was applied to the sections for a minimum of 30 min, followed by streptavidin-horseradish peroxidase for an additional period of at least 30 min. Diaminobenzidine was used as the chromogen, and the sections were lightly counterstained with hematoxylin before coverslipping. Negative control slides, consisting of sections incubated overnight with diluent only or normal rabbit serum at 1:200 in diluent, rather than the primary antibody were performed, and a case known to give a positive result was included in each staining run.
Semiquantitative analysis of each section was carried out. The number of tumor cells stained positively was counted, and the percentage of cells stained was calculated for each case. Tumor cell counting was facilitated by viewing sections and counting with a video image analysis system (Optimas Corp., Seattle, WA). In accordance with some other reports (21, 23), tumors with 10% or more of cells staining positively were designated positive.
Extraction of RNA
Frozen tumor tissue stored at -70 C for up to 6 yr was pulverized in a supercooled vessel, suspended in a guanidinium isothiocyanate solution, and homogenized using a Dounce homogenizer (Kontes Co., Vineland, NJ) and Teflon pestle. Total RNA was extracted by the guanidinium isothiocyanate-cesium chloride method as previously described (24).
RT-PCR amplification of ER and PR transcripts
Fragments of the ER transcript were amplified by the RT-PCR
using the primers indicated in Fig. 1
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Oligonucleotide sequences are given below. Details of the ER sequence
are taken from the reports of Greene et al. (25) and
Ponglikitmongkol et al. (26). An EcoRI
restriction site was added to the 5'-end of some sense primers, and
this is indicated in bold type. Sequences for primers ER 1, ER 2 and ER
D were taken from the report by Fuqua et al. (27): ER
A, GGAATTC AGC CCG CTG ATG CTA CTG (ER 316–333); ER B, TCA
TCA TTC CCA CTT CGT AGC (ER 734–754); ER C, GGAATTC TGC TTC
AGG CTA CCA TTA TGG (ER 573–593); ER D, TGA ACC AGC TCC CTG TCT GCC
AGG TTG GT (ER 1039–1067); ER E, GGAATTC AAA AAC AGG AGG
AAG AGC TGC (ER 691–711); ER F, GCA AAC AGT AGC TTC CCT GGG (ER
1194–1214); ER F2, CAG GAT CTC TAG CCA GGC AC (ER 1142–1161); ER 1,
GGA GAC ATG AGA GCT GCC AAC (ER 850–870); ER 2, CCA GCA GCA TGT CGA
AGA TC (ER 1269–1288); ER G, GGAATTC CCT TCT AGA ATG TGC
CTG GC (ER 1131–1150); ER H, TTC TCT TCC AGA GAC TTC AGG G (ER
1394–1415); ER 6, TAG AGG GCA TGG TGG AGA TC (ER 1253–1272); ER 8,
CTT CAT GCT GTA CAG ATG CTC C (ER 1566–1587); and ER 8.2, GTA ACA AAG
GCA T (ER 1553–1565).
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Southern blot analysis
Aliquots of each PCR product mix were electrophoresed through agarose gels containing ethidium bromide and were transferred to nylon membranes, (Hybond N+, Amersham, Castle Hill, Australia), using capillary action. Membranes were probed with exon-specific oligonucleotide sequences from ER and PR and PR cDNA, labeled with 32P. Radioactively labeled membranes were evaluated using a Molecular Dynamics, Inc., PhosphorImager and ImageQuant software (Sunny- vale, CA).
A semiquantitative estimate of alternatively spliced transcript
expression was made by comparing the intensities of the variant and
wild-type bands on Southern blots as previously described (21, 28).
Levels of the variant transcripts were expressed as a percentage of the
wild-type level. Levels of the 5ER reported in Table 1
are derived from the mean results of 2 separate determinations for each
tumor. Relative expression of the most abundant variant, the
7ER, was examined in detail. For this
reaction, the kinetics of amplification were documented by comparing
the intensity of the wild-type and 7ER signals
on Southern blot over a range of PCR cycles. These results showed that
both products were amplified with similar efficiency, and the ratio of
the two products was comparable throughout the reaction (data not
shown). Semiquantitative results of the relative expression of
7ER are the mean values from 3 separate RT-PCR
reactions for 30 tumors and from 2 reactions for 5 cases. In each case,
measurements were made in both the plateau (30 cycles) and linear (19
cycles) phases of amplification.
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Sequence verification of the 7ER,
4PR, and 6PR variants
was performed by subcloning the RT-PCR products into the pGEM-T vector
(Promega Corp., Sydney, Australia). The
4/2PR variant was sequenced directly from the
RT-PCR product. Sequencing was performed by automated dideoxy chain
termination sequencing (373A DNA Sequencing System, Applied Biosystems,
Foster City, CA).
Statistical methods
Statistical tests were performed using Abacus Concepts StatView Student software (Abacus Concepts, Inc., Berkeley, CA).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Segments of the ER transcript spanning each of exons 2–7 were
separately amplified by RT-PCR. Examples of Southern blots from these
experiments are shown in Fig. 2. A region
spanning exon 2 was amplified, and a product corresponding in size to
the expected 446-bp wild-type ER was seen (Fig. 2A). This band
hybridized with an oligonucleotide positioned in exon 3 (ER E) on
Southern blots, and in addition, a smaller product corresponding in
size to an ER transcript lacking exon 2 (2ER) was
revealed in all cases. Further evidence of the identity of the variant
was derived from an experiment in which a duplicate of one of the
Southern blots was probed with an oligonucleotide from the exon 2 ER
sequence (ER C), and the smaller band failed to hybridize (not
shown).
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). A smaller band was only faintly seen in some cases. To
visualize products more clearly, RT-PCR products from a subset of
cases were run on 6% polyacrylamide gels and stained with silver. By
this method, a band of approximately the correct size for the exon
3-deleted ER variant (3ER) was seen, which may represent
a variant ER transcript, however, the intensity of this band was very
low compared with that of wild-type ER (data not shown). It was
concluded that the 3ER was not confidently identified in
this series, and if present, the level of expression was likely to be
very low.
The predicted exon 4-deleted ER transcript
(4ER) was seen in addition to wild-type ER on
Southern blots probed with an exon 5 ER sequence (ER F2; Fig. 2C) in 33
of the 35 cases, and when 1 of the blots was stripped and reprobed with
an oligonucleotide in exon 4 (ER 1), the variant was not detected (not
shown). Primers used in the original description of the exon 5-deleted
ER variant (5ER) were used to amplify the ER
sequence spanning exon 5 (27) (Fig. 2D). The
5ER, identified on the basis of its size and
differential hybridization to an oligonucleotide probe in exon 4 (ER D)
but not one in exon 5 (ER F2; not shown), was detected in 29 of the 35
tumors. Amplification of exon 6 revealed no evidence of an exon
6-deleted variant (6ER) in any of the cases on
Southern blots (Fig. 2E) or when a subset was run on acrylamide gels
and stained with silver (data not shown).
In all of the ER-positive cases, amplification across exon 7 revealed
wild-type and the exon 7-deleted variant
(7ER). In contrast to the other variants, the
7ER could be discerned on agarose gels
containing ethidium bromide; consistent with this, it was the most
intense signal relative to that of the wild type on Southern blots
(Fig. 2F). The variant hybridized with ER sequences from exons 6 (ER 2)
and 8 (ER 8.2), but not a probe positioned in exon 7 (ER H) (not
shown). The identity of the variant was further proven by subcloning
and sequencing of the wild-type and 7ER RT PCR
products from two tumors (not shown).
Expression of alternatively spliced PR transcripts
Of the 35 ER-positive tumors examined, 26 were PR positive by EIA.
The PR transcript was examined in 25 of these by amplifying 2 fragments
by RT-PCR: one spanning exons 2, 3, and 4 and the other spanning exons
5, 6, and 7, (16). Using this strategy, a number of variant PR
transcripts were detected in addition to wild-type PR. Southern blots
from these experiments are illustrated in Fig. 3.
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), which, on the basis of size and the pattern of hybridization to
oligonucleotide probes positioned in exons 2 and 3, are likely to
represent variant PR transcripts from which exon 2 (2PR)
and both exons 2 and 3 (2+3PR) were deleted. On
Southern blots, each of these variants produced only a faint signal,
which was not clearly discerned in all cases. A PR variant from which
exon 4 of the PR sequence had been deleted
(4PR) was identified in 24 of the 25 cases.
The identity of this species was confirmed by the pattern of
differential hybridization to oligonucleotide probes; in addition, the
4PR variant was subcloned and sequenced from 3
tumors (data not shown). A product of approximately 600 bp was also
seen in these experiments. Differential hybridization with
oligonucleotides in exons 3 and 4 of PR revealed that 2 separate
species were contributing to this band: a product from which exon 3 of
the PR sequence was deleted (3PR) and a novel variant
from which the first 126 bp of exon 4 were deleted
(4/2PR; Fig. 3C).
When a fragment of PR spanning exons 5, 6, and 7 was amplified (Fig. 3B), a product of 657 bp was identified. When this was subcloned and
sequenced, it was proven to be a PR variant from which the exon 6
sequence was deleted (6PR). In addition, a
faint band corresponding in size to a transcript lacking both exon 5
and 6 sequences (5+6PR) was noted. No transcripts
lacking exon 7 of PR were detected.
Relative expression of ER and PR alternatively spliced transcripts
When the level of expression of variant transcripts was compared
semiquantitatively with that of the wild-type ER or PR transcript, it
was found that for each variant there was a characteristic level of
expression, and the range for the tumors examined was quite small
(Table 1). In general, ER variant
transcripts were more abundant than PR variants.
The most highly expressed species was the 7ER,
which was detected at levels ranging from 29–83% of wild-type ER
(mean, 51%). Mean levels of expression of the other ER variants were
less than 25% that of wild type. Of the PR variant transcripts, the
most abundant species was the 6PR, which was
detected in 23 of 25 tumors and had a mean level of expression of 8%
that of wild-type PR. The other 2 relatively abundant PR variant
species were 4PR and
4/2PR, which had mean levels of expression of 2%
and 4% that of the wild type, respectively.
For each alternatively spliced transcript species detected, there were individual tumors that contained high levels compared with the overall population; however, a general propensity to form alternatively spliced transcripts was not seen as these cases did not necessarily express high levels of other variant species (data not shown).
Coexpression of alternatively spliced ER and PR transcripts and expression of the estrogen-responsive proteins PR and pS2
To determine whether the expression of variant transcripts was
related to markers of hormonal sensitivity in breast cancers, the
profile of expression of the estrogen-responsive proteins, PR and pS2,
was examined in the 35 primary breast cancer specimens (Table 2). All of the tumors were ER positive by
EIA, except for a single case with ER of 4 fmol/mg protein but ER
positive by immunohistochemistry which was therefore included. In this
small cohort there was considerable heterogeneity in the expression of
the two proteins, with 51% being PR+pS2+, 14%
being PR-pS2+, 23% being
PR+pS2-, and 11% being
PR-pS2-.
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2ER and 7ER, and none
contained the 3ER and 6ER
variants. The 4ER was absent from 2 cases
only. The 5ER was present in 29 of the 35
cases. Three of the 6 cases in which the variant was not identified
contained both PR and pS2; one was PR-pS2+,
one was PR+pS2-, and the remaining case
expressed neither protein. The distribution with respect to PR and pS2
was similar for cases that contained the 5ER
as for those that did not. It was clear, therefore, that there was no
relationship between the profile of ER variants and receptor
function.
The level of expression of the most abundant variant,
7ER, was compared with the expression of PR
and pS2. When the cases were subdivided according to the pattern of
expression of PR and pS2, no relationship with the
7ER level was seen (by one-factor ANOVA,
P = 0.19; Fig. 4).
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3PR, 4PR,
4/2PR, and 6PR species
were detected in the majority of tumors. The
2PR and transcripts lacking two exons
(2+3PR and 5+6PR) were detected
infrequently (Table 2); however, it was notable that these latter
transcripts were present in very low abundance, so it cannot be
excluded that failure to detect these in some tumors may be related to
the limit of detection of the method.
When expression of the most abundant alternatively spliced PR
transcripts (4PR, 4/2PR,
and 6PR) was compared on the basis of pS2
positivity, no difference was noted between pS2+ and
pS2- tumors (Table 2).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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2ER and
7ER variant transcripts, whereas
3ER and 6ER variants
were not seen. The 4ER was absent from two
cases only. The 5ER was detected in the
majority of tumors, but there was no apparent relationship between
expression of the 5ER and the pattern of
expression of PR and pS2. There was no concordance between the small
groups of cases in which 4ER and
5ER were not found. It is apparent, therefore,
that there is a common profile of variant ER transcripts in breast
cancer, and this feature is not related to receptor function, as
revealed by the presence of estrogen-dependent end points. These data
are consistent with results reported by Zhang et al.
(18).
A subset of 25 tumors from this cohort that were both ER and PR
positive were examined using a similar strategy for the presence of
variant PR transcripts. Multiple truncated PR transcripts were detected
in all cases. The 3PR,
4PR, 6PR, and
4/2PR species were detected in almost all tumors.
There was a single PR+pS2+ case in which
4PR, 6PR, and
4/2PR were not seen and two other cases that failed
to express either 3PR or 6PR.
The level of expression of the other less commonly detected variants,
2PR, 2+3PR, and
5+6PR, was very low, and it is likely therefore
that their apparent absence from some cases is due to their being below
the level of detection.
These data suggest that primary breast tumors are not distinguished by the profile of expression of ER or PR variant transcripts and that this feature cannot, therefore, be indicative of hormone responsiveness. Further, a general propensity for alternative splicing of ER and PR transcripts was not seen, as cases that expressed high levels of a specific variant relative to other members of the cohort did not necessarily contain high levels of others.
Alternative splicing of the primary transcript is the most likely
mechanism for the formation of variant transcripts from which entire
exon sequences have been deleted. With respect to both ER and PR, the
multiplicity of variants detected and the distinct profiles of these
indicate that the primary transcripts are subject to a number of
alternative splicing events, and there are specific splice junctions
that are vulnerable to this process. It is noted, for example that
although exons 2, 4, 5, and 7 were commonly excluded from ER, a variant
ER transcript lacking exon 6 was not detected in this series and has
never been identified. Similarly for PR, deletion of exons 2, 3, 4, and
6 were observed, whereas an exon 7-deleted variant was not seen, and
exon 5 was deleted only in combination with exon 6. Insight into the
selection of splice sites may come from the novel PR variant
4/2PR that was detected in this series. This
transcript is the result of deletion of 126 bp from exon 4 of the PR
sequence and is likely to be formed by the splicing together of the
native donor splice site from the 3'-end of PR exon 3 with a cryptic
acceptor site within exon 4 at position 2776 (29). It is noted that the
bases bounding this cryptic splice junction conform to the acceptor
splice site consensus sequence (Table 3),
suggesting that the formation of the 4/2PR variant
may be due to the cryptic splice site successfully competing with the
native sequence for recognition in the splicing reaction in a small
proportion of transcripts. In the formation of others of the
alternatively spliced ER and PR variants, however, the degree of
conformation to the consensus sequence of splice sites that are never
excluded in the formation of variant transcripts is not greater than
those that are, suggesting that factors other than splice site strength
are likely to determine the pattern of alternative splicing.
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5ER by immunoprecipitation followed by
immunoblotting in the BT20 breast cancer cell line (32), and Desai
et al. using an anti-5ER monoclonal
antibody reported specific staining in frozen sections from two breast
tumors (10). In addition, Park et al. demonstrated the
4ER protein on Western blots of ovarian
carcinoma and other normal tissues (14), and Erenburg et al.
reported low level expression of a protein corresponding to the
3ER in the MCF-7 breast cancer cell line (13).
Candidate variant PR proteins that may be encoded by the alternatively
spliced PR transcripts have not been specifically demonstrated. A
truncated PR protein seen in 25% of PR-positive breast tumors on
Western blot is unlikely to correspond to one of these variants (16);
however, other small immunoreactive bands seen in some tumors on these
blots are potential candidates (22).
In vitro studies of variant receptor function have supported
the view that these variants may impact on hormone sensitivity;
however, this has not been confirmed to date by studies of clinical
material. One reason for this discrepancy might be that the effects
demonstrated in vitro have been in the context of much
higher relative expression of the variants than are present in
vivo. Wang et al. reported that cotransfection of a
20-fold excess of 2ER to wild-type ER in HeLa
cells resulted in mild inhibition of ER function, and when
3ER was expressed at an equivalent level as
wild-type ER, a 30% reduction in ER activity was noted (12). With
respect to PR variants, Richer et al. reported that a
10-fold excess of 6PR reduced the activity of
PR B by 60–70% and equimolar 5+6PR reduced the
activity of PR B by 20% and that of PR A by 65% (17). In this study,
however, the mean level of expression of 2ER
transcript was 18% that of wild-type ER, and the mean level of
expression of 6PR was 8% that of wild-type
PR. The 3ER and 5+6PR
variant transcripts were barely detectable. Although results derived
from RNA extracted from a tissue homogenate are likely to be a crude
reflection only of events at a molecular level, the low level of
relative expression of the alternatively spliced transcripts in breast
tumors imply that corresponding variants would require more powerful
dominant activity than has been reported to date to significantly
impact on hormone sensitivity.
The most abundant variant species detected in this series was the
7ER, which was present at levels ranging from
29–83% of wild-type levels. In this range of expression, an effect on
ER function might be expected, as Fuqua et al. reported a
60% reduction in ER function in a yeast expression system when the
7ER was expressed at an equivalent level as
wild-type ER (11). In contrast to reports that the
7ER was more highly expressed in
ER+PR- tumors than
ER+PR+ cases (11, 18), in this study the
relative level of the 7ER could not be related
to the expression of the estrogen-responsive proteins PR and pS2, and
there was no evidence therefore to implicate the
7ER in aberrant ER function.
In summary, we have found in a series of 35 primary breast tumors that multiple alternatively spliced ER and PR transcripts coexist and that there is a common profile of these variants. This uniformity is in contrast to considerable variation in receptor levels and expression hormone-responsive markers and implies that alternative splicing of the primary transcript may not be a determinant of receptor function.
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Acknowledgments |
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Footnotes |
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2 R.L.B. was a National Health and Medical Research Council Medical
Postgraduate Scholar.
Received October 27, 1998.
Revised January 12, 1999.
Accepted January 20, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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3) in breast cancer and the consequences
of its reexpression: interference with estrogen-stimulated properties
of malignant transformation. Mol Endocrinol. 11:2004–2015.This article has been cited by other articles:
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  A. A. Iverson, C. Gillett, P. Cane, C. D. Santini, T. M. Vess, L. Kam-Morgan, A. Wang, M. Eisenberg, C. M. Rowland, J. J. Hessling, et al. A Single-Tube Quantitative Assay for mRNA Levels of Hormonal and Growth Factor Receptors in Breast Cancer Specimens J. Mol. Diagn., March 1, 2009; 11(2): 117 - 130. [Abstract] [Full Text] [PDF]
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