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Tumor Biology |
University Departments of Surgery [A. G., P. A. H., N. J. B.] and Pathology [W. F. K.], University Hospital of South Manchester, Manchester M20 2LR, and Department of Epithelial Biology, Cancer Research Campaign Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 4BX [C. S. P.], United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSHormonal Manipulation of...RESULTSApoptosis (Fig. 1
)Cell Proliferation (Fig. 2...DISCUSSIONREFERENCES |
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Women (n = 23) with mammographic microcalcification suggestive of DCIS were identified at the time of surgery (day 0), a sample of representative tissue was obtained, divided into multiple 2 x 2 x 1-mm xenografts, and implanted s.c. into female BALB/c nu/nu mice (eight xenografts/mouse). Day 0 grafts underwent immunohistochemical assessment of ER status. Fourteen days after implantation, four xenografts were retrieved and mice were randomly divided into one of three treatment groups: (a) insertion of a slow release 2-mg 17ß-estradiol pellet; (b) weekly 5-mg injections of the pure AE Faslodex (Zeneca Pharmaceuticals); and (c) injections of a control vehicle oil alone. After 2 weeks of treatment, the remaining four xenografts were retrieved from each mouse. Retrieved xenografts containing DCIS were assessed for morphological evidence of apoptotic cell death [apoptotic index (AI)] and cell proliferation (by immunohistochemical detection of the Ki67 proliferation antigen LI).
Both AI and LI were higher in the day 0 specimens of 16 ER- DCIS lesions compared with 7 ER+ DCIS lesions (mean values, 1.47% versus 0.32% and 20.6% versus 3.1%; both P < 0.0001). AI and LI values within ER- DCIS did not differ between xenografts exposed to 17ß-estradiol or AE treatment compared with the controls or pretreatment values (mean AI and LI in estradiol-treated, antiestrogen-treated, and control groups 1.04% versus 0.98% versus 1.29% and 17.2% versus 20.5% versus 17.7% respectively). In contrast, treatment of mice bearing ER+ DCIS xenografts with 17ß-estradiol raised both the AI (1.03% versus 0.40%, P = 0.03) and LI (11.0% versus 5.1%, P = 0.007) compared with controls. AE therapy of ER+ DCIS xenografts did not affect proliferation but resulted in higher apoptosis than in controls (0.9% versus 0.4% respectively, P = 0.04).
AE therapy should be reserved for patients with estrogen receptor-positive DCIS.
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INTRODUCTION |
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The NSABP group has recently published data examining the role of the AE tamoxifen in the treatment of DCIS following breast-conserving surgery and localized radiotherapy (3) . Tamoxifen reduced the incidence of ipsilateral invasive breast cancer recurrence by 44%, and they recommended that adjuvant tamoxifen be used for all women undergoing breast-conserving surgery for DCIS. However, no information regarding ER status of tumors was given in this study. In women with ER- invasive breast carcinoma, tamoxifen has been shown to provide little benefit (4 , 5) , and our recently published in vivo model has demonstrated that ER- DCIS is, likewise, hormone independent (6) . In the NSABP B-24 trial, one in three women were reported unable to complete the course of tamoxifen treatment due to a combination of unacceptable effects of the drug or "personal reasons." There was also an excess of endometrial cancer and thromboembolic events in the treated group compared with controls (3) . In women with unresponsive DCIS such adverse effects of tamoxifen assume greater clinical concern.
Although in vitro studies have shown a definite apoptotic effect of AE therapy on both ER+ and ER- cell lines (7 , 8) , the clinical relevance of these studies are unknown. Additional studies of the effects of AEs on DCIS at the cellular level may aid distinction between DCIS lesions likely to respond, or not, to hormonal therapy and would potentially be clinically useful. The absence of cell lines for DCIS led us to develop an in vivo animal model in which human DCIS xenografts could be subjected to hormonal manipulation (6) . We showed that oestrodiol exposure generated increased cell proliferation in ER+, but not ER-, DCIS (6) and hypothesized that proliferative drive in ER- DCIS was unlikely to be controlled by estrogen or AE therapy. To test this hypothesis, we have used the same model to expose human DCIS xenografts to pure AE therapy and examined the effects on rates of cell proliferation and apoptotic cell death. We show that neither cell proliferation nor apoptotic cell death is affected by AE exposure in ER- DCIS, in direct contrast to ER+ DCIS.
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MATERIALS AND METHODS |
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Treatment of Tissue Samples.
A small (1–2 cm3
) sample of representative
tissue was immediately removed from the surgically excised specimen,
stripped of excess fat, placed in DMEM with 4.5 g/liters of glucose and
without sodium pyruvate (Life Technologies, Inc., Paisley, Scotland) at
room temperature, and transported to the laboratory without delay. The
tissue was then placed in fresh DMEM in a Petri dish and dissected into
2 x 2 x 1-mm xenografts using a scalpel
blade. Depending on the volume of tissue available, between 10 and 100
xenografts were randomly picked from the Petri dish and fixed with 4%
formaldehyde solution for 24 h, followed by storage in 70%
alcohol until paraffin embedding. These samples, representing the DCIS
excised from each patient, were labeled as "day 0 specimens" and
were reserved for histological review, immunostaining, and apoptotic
cell counts.
The remaining xenografts were implanted into nude mice for hormonal manipulation.
Animals.
Intact, female athymic nude mice (BALB/c nu/nu), 9–10 weeks of age,
were obtained from the breeding colony at the Paterson Institute for
Cancer Research. The mice were housed under conventional conditions
with a 12-h cycle of light and dark (lights off 1900–0700 h) in filter
top cages and supplied ad libitum with normal feed, water,
and bedding during the experiments. Irradiated bedding, irradiated
food, and filtered water were used during production (breeding). Animal
care and all surgical procedures were performed in strict accordance
with Home Office Regulations and United Kingdom Scientific Procedures
(1986) Act.
Implantation of DCIS into Nude Mice.
Each patient’s sample was divided and implanted into 
5–24 (median,
14) mice depending on the volume of tissue available. Transplantation
of xenografts into the nude mice was completed within 90 min of removal
of tissue from the patient. Two small midline skin incisions were made
across the dorsal skin through which eight xenografts were
symmetrically placed (two by each scapula and two by each hindquarter).
Halothane inhalation anesthesia was used for each procedure (2–4%
halothane in oxygen; Halovet Vaporiser, International Market Supplies,
Congleton, United Kingdom).
Removal of xenografts involved reanesthetizing the mice and use of sharp dissection for excision of grafts at the appropriate time point. On removal from the mice, each graft was immediately fixed in 4% formaldehyde solution for 24 h, followed by storage in 70% alcohol until paraffin embedding.
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Hormonal Manipulation of Xenografts |
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Estrogen Administration.
A s.c. tunnel was made to the base of the tail to allow the placement
of a slow release silastic encapsulated estrogen pellet containing 2 mg
of 17ß-estradiol (E-8875; Sigma Chemical Co., St. Louis, MO). The
preparation, use, and effects on serum estradiol of these pellets has
been previously described in detail (5
, 9)
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AE Administration.
The AE used was Faslodex [7
-[9-(4,
4,5,5,5-pentaflouropentylsulfinyl)nonyl]estra-1,3,5,
(10)
-triene-3,17ß-diol), previously known as ICI 182 780], kindly
donated by Dr. A. Wakeling (Zeneca Pharmaceuticals, Cheshire, United
Kingdom). This compound has pure antiestrogenic and no partial estrogen
agonist properties both in vivo and in vitro
(10)
and has also been shown to have potent antiestrogenic
effects in a nude mouse model using breast cancer cell lines as
xenografts (11)
. Five milligrams of Faslodex suspended in
1 ml of propylene glycol-based vehicle oil was injected s.c. into the
nape of the neck (without the use of anesthetic) in the appropriate
mice 14 and 21 days after implantation of the xenografts.
Control Group.
Control mice received 1 ml of vehicle oil injections injected in a
similar fashion to AE on days 14 and 21.
After the 14-day treatment period, four remaining xenografts were retrieved from each mouse.
Histological Evaluation of Xenografts.
All day 0 specimens and each xenograft were embedded into
paraffin blocks. H&E-stained, 3-µm sections from each block were
examined by a single experienced breast pathologist (W. F. K.)
for the presence of DCIS; those containing DCIS were assessed for
apoptosis and Ki67 antigen immunogenicity (as a marker for cell
proliferation). In addition, day 0 specimens were evaluated
immunohistochemically for ER status.
For each experiment, the number of day 0 specimens containing DCIS was used as an indication of the proportion of the xenografts expected to contain DCIS tissue. For example, in an experiment in which 33% of the day 0 specimens contained DCIS, then one in three retrieved xenografts would be expected to return DCIS. However, if only one in six retrieved xenografts contained DCIS, then a 50% return would be recorded
Assessment of Apoptotic Cell Death.
H&E-stained sections of DCIS samples were examined using light
microscopy for morphological evidence of apoptosis. The criteria used
to identify apoptotic cells are well recognized (12
, 13)
and include condensation of chromatin initially at the margins of the
nucleus; condensation of the cytoplasm (chromophilia); detachment from
surrounding cells, indicated by the appearance of a characteristic halo
around the dying cell; and cytoplasmic budding to form membrane-bound
fragments (apoptotic bodies).
To obtain the AI, a minimum of 500 cells (1000 cells for day 0 specimens) were counted, using a x40 Planapo oil lens and a Zeiss microscope, and the number of cells displaying apoptotic morphology were expressed as a percentage of the total number counted, as described previously (14) .
Immunohistochemical Determination of Ki67 Nuclear Antigen.
A standard three-layered streptavidin-avidin-biotin horseradish
peroxidase method was used with diaminobenzidine (DAKO Ltd., High
Wycombe, United Kingdom) as the chromogen. The primary antibody
was a polyclonal rabbit antihuman Ki67 antigen (DAKO A047, 1:50
dilution). A swine antirabbit biotin-labeled polypeptide (DAKO E431,
1:400 dilution) was used as the secondary antibody.
Ki67 immunostaining was predominantly nuclear with little cytoplasmic uptake. The intensity of staining was variable, but this was not assessed separately, and the cells were judged as positive or negative. Ki67 and apoptosis assessment of retrieved xenografts were performed by investigators (A. G., W. F. K.) blinded to the treatment group.
Assessment of ER Status.
A standard three-layered streptavidin-avidin-biotin horseradish
peroxidase method was used with a mouse antihuman ER primary antibody
(DAKO M7047, 1:100 dilution) and a biotinylated rabbit antimouse
secondary antibody (DAKO E413, 1:350 dilution). Staining was
predominantly nuclear with little or no cytoplasmic staining. A minimum
of 1000 malignant cells were counted, and lesions were considered ER+
if >5% of cells were positively stained for ER.
Assessment of PR Status.
Day 28 xenograft sections from ER+ experiments were labeled for
PR using serum from the PR-ICA kit (Abbot Diagnostics). A standard
three-layered streptavidin-avidin-biotin horseradish peroxidase method
was used with an overnight application of rat monoclonal primary
antibody (1:4 dilution) and use of a biotinylated rabbit antirat
secondary antibody at 1:100 dilution. Similar to ER, staining was
predominantly nuclear with little or no cytoplasmic staining. A minimum
of 500 cells were assessed, and positively stained nuclei were
expressed as a percentage of the total number counted.
Statistical Methods.
For each DCIS case, a comparison was made between DCIS samples
retrieved from the three treatment groups and also between the three
different time points [day 0, day 14 (pretreatment), day 28
(treated)]. The tissue samples obtained at each assessment day for the
three study groups were all considered statistically independent.
Differences between the groups and between the assessment days were
evaluated by use of ANOVA. Both AI and LI were found to follow a skewed
non-Normal distribution and were converted to natural logarithms for
analysis. Transformation to loge gave an adequate
approximation to Normality for all analysis in the model. The results
obtained were then detransformed into the original units for
presentation and are presented as geometric means and their 95% CIs.
Pairwise comparisons indicated by the f-ratios from the ANOVA were
investigated by Tukeys’ multiple comparison test. Association between
AI and LI in day 0 samples was explored using the Pearson correlation
following transformation to loge and confirmation
by scatterplot of a robust linear relationship.
Comparisons of the day 0 results between ER- and ER+ samples and between samples displaying some or no comedo-necrosis were carried out using simple ANOVA, as were comparisons of mouse weights, which are presented as the true (arithmetic) means and their 95% CIs for each treatment group.
All significance tests were two-sided and used the conventional 5% significance level.
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RESULTS |
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Histopathology of Nonimplanted Day 0 Specimens.
Sixteen of the 23 experiments (70%) contained ER- DCIS, and 7 cases
(30%) contained ER+ DCIS (median number of cells staining positive for
ER, 53%; range, 22–82%). Eleven (69%) of the 16 ER- women and 2
(29%) of the 7 ER+ women presented symptomatically. In only one case
did the ER status of the xenografts not correlate with that of the
histological slides (i.e., there was 95% concordance
between ER status of day 0 xenografts and clinical tissue samples from
which day 0 tissue was obtained). The DCIS in 17 women (74%) was
designated as high grade, and three each (13%) as intermediate and low
grade. According to the criteria of Page et al.
(15)
, one of seven of the oestrogen receptor-positive DCIS
samples and 15 of 16 of the oestrogen receptor-negative DCIS samples
were of the comedo histological subtype, all of which demonstrated
evidence of comedo-necrosis, thus displaying a significant association
between ER status and presence of comedo-necrosis
(P < 0.001, degrees of freedom = 1; two-sided Fishers exact test). The remaining seven samples
were all of the noncomedo variety, none of which showed evidence of
necrosis. The mean AI was higher in those tissue samples displaying
evidence of necrosis than in those without necrosis (mean AI, 1.34%
versus 0.43%; f[1.35] = 19.9,
P < 0.001). A clear association between
comedo-necrosis and raised AI has been noted previously (14
, 16)
.
The 23 breast biopsies gave rise to a total of 546 day 0 specimens (i.e., nonimplanted xenografts), of which 151 (28%) contained foci of DCIS (median number containing DCIS, 24%; range, 7- 67%).
Overall DCIS Retrieval from Xenografts.
The 23 experiments produced a total of 2444 xenografts, of which 2321
(95%) were retrieved (median value for each experiment, 97%; range,
50–100%). Of these, 763 (33%) contained foci of DCIS.
The median retrieval of DCIS per experiment was 92% of that expected (range, 33–203%). DCIS was retrieved from day 14 xenografts in 91% (21 experiments) and from day 28 xenografts in 100% of the 23 experiments.
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Apoptosis (Fig. 1
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Cell Proliferation (Fig. 2
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSHormonal Manipulation of...RESULTSApoptosis (Fig. 1
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A positive correlation was seen between apoptotic and proliferative indices in all day 0 specimens (Pearson correlation coefficient, r = 0.5, P = 0.02).
The antiestrogenic effects of Faslodex on ER+ DCIS xenografts was confirmed by immunohistochemical assessment of PR within day 28 xenografts. Expression of the PR protein, an estrogen-regulated gene product (17) , was reduced in Faslodex-treated xenografts [geometric mean PR score, 18.17% (95% CI, 17.99–18.24%)] compared with control xenografts (mean PR score, 43.38% (95% CI, 34.12–54.6%)], estrogen-treated xenografts [mean PR score, 46.53% (95% CI, 41.68–51.94%)], or day 0 specimens [mean PR score, 39.25% (95% CI, 32.14–47.94%)]; f[3,14] = 18.6, P < 0.001.
Mouse Weights.
Mean mouse weights at day 0 were 24.0 g (95% CI, 23.6–24.5 g),
increasing to 25.0 g (95% CI, 24.4–25.6 g; f[1.38] = 6.8, P = 0.01) on day 14 when treatment was
commenced. There was no difference in the weight of mice treated by AE
injections [mean weight, 25.7 g (95% CI, 24.0–26.8 g)]
compared with the control group [mean weight, 25.8 g (95% CI,
24.9–26.0 g)], but was raised by treatment with an estradiol implant
[mean weight, 28.0 g (95% CI, 26.3–29.7 g); f[2,38] = 6.6, P = 0.004].
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSHormonal Manipulation of...RESULTSApoptosis (Fig. 1
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Overall xenograft survival was excellent with 95% of all implanted grafts successfully retrieved. One in three retrieved xenografts contained DCIS for analysis, and the median DCIS retrieval per experiment was 92% of that predicted from the day 0 specimens.
The AE Faslodex (previously known as ICI 182780) was used because this agent displays pure antiestrogenic properties (10) with none of the partial estrogen agonist properties associated with tamoxifen (18) . Previous studies have shown the superior antitumorigenesis properties of Faslodex over tamoxifen in MCF-7 cell line tumors raised in nude mice (11) . The precise mechanism of the antitumor activity exhibited by AE compounds is not known, but two important pathways have emerged; a reduction in cell proliferation and/or an increase in apoptotic cell death within the tumor mass.
AE Effects on Proliferation.
Immunohistochemical detection of Ki67 nuclear antigen has been shown to
closely correlate with other techniques of cell proliferation
assessment (14
, 19)
and is widely used to monitor tumor
proliferation in response to various treatments. We examined the
effects of pure AE treatment on Ki67 immunostaining within DCIS
xenografts.
ER- DCIS displayed a significantly higher resting cell proliferation rate than ER+ DCIS and showed no change following 14 days of either estradiol or AE therapy. In contrast, ER+ DCIS xenografts displayed a marked increase in the Ki67 proliferation index when subjected to 14 days of estradiol exposure, although AE treatment had no effect on cell proliferation. In vitro studies of the growth inhibitory properties of Faslodex have shown a cytostatic effect with accumulation of cells in the G0/G1 phase (10) . If this same effect were seen in vivo, then a fall in the LI would be expected in ER+ DCIS xenografts exposed to AEs. The lack of such a finding in our model may be due to the short period of AE treatment or to the dose of AE used (based on work using a similar mouse model; Ref. 11 ). However, Brünner et al. (20) found that the effects of AE treatment of ER+ MCF-7 cells in vitro resulted in cell accumulation in the G1 phase of the cell cycle (i.e., cytostatic effect), but treatment in an in vivo model (MCF-7 cell xenografts) had no effect on cell cycle kinetics. Nevertheless, AE treatment of in vivo MCF-7 tumors resulted in tumor regression, and Brünner et al. (20) suggested that, in the absence of changes in proliferation kinetics, tumor shrinkage must be due to an increase in cell loss.
AE Effects on Apoptosis.
Several studies, in vitro and in vivo, have
demonstrated that apoptotic cell death can be induced in breast cancer
cells by estrogen withdrawal or by AE administration (21
, 22)
. Cells exhibiting apoptosis display a characteristic
morphology (12
, 13) that may be recognized by light
microscopy. We have used these properties to determine an AI for each
xenograft containing DCIS, thus allowing comparisons between treatment
groups.
ER- DCIS had a high resting AI compared with ER+ DCIS and remained unaltered after14 days of treatment with either estradiol or AE therapy. In contrast, ER+ DCIS xenografts showed a marked increase in apoptotic cell death when subject to either estradiol or AE exposure compared with pretreatment and control groups. Whereas estradiol treatment resulted in an accompanying rise in cell proliferation, no such effect was seen following AE treatment. Previous in vivo studies showed no effect of AE on cell cycle distribution (i.e., no antiproliferative effect) in MCF-7 cell xenografts, yet tumor regression was induced (20) . Our data, using human DCIS xenografts, suggests that this regression is due to induction of the apoptotic process.
Other methods for identifying apoptosis include ISEL and terminal deoxynucleotidyl transferase-mediated nick end labeling. These processes are thought to simplify detection of apoptotic cells by incorporating biotinylated deoxyuridine at DNA strand breaks within apoptotic nuclei. However, doubts over the specificity of these end labeling techniques in distinguishing between apoptotic and necrotic cell death are well documented. ISEL detection of DNA strand breaks is not specific for apoptotic cell death and can be positive in cells that show no evidence of apoptosis (23) , as well failing to stain cells showing quite classical features of programmed cell death (24) . Both the ISEL and terminal deoxynucleotidyl transferase-mediated nick end labeling assays can fail to discriminate between apoptotic and necrotic cell death (23 , 25) , and there may be a discrepancy between the two staining techniques in the detection of apoptosis in breast cancer tissue (26) . In our own laboratory, we have noted that ISEL staining gives false positive rates of 0.3–1.6% and false negative rates of 17.3- 35% (27) . While accepting that morphological assessment of apoptotic counts may detect only cells in the latter stages of the apoptotic process, we consider that light microscopic identification of cells displaying the recognized morphology of apoptosis is the most reliable and reproducible method of evaluating apoptotic cell counts (28) .
Decreased detection of estrogen-regulated gene products following treatment of breast cancer cells with pure AEs has been documented previously and is thought to be due to the attenuated transcriptional activity of the pure AE-ER complex (11) . Our model clearly demonstrated the negative effects of Faslodex on immunohistochemical expression of the estrogen-regulated PR protein (18) within oestrogen receptor-positive DCIS xenografts. This finding has also been noted in human subjects (29) using the same AE and verifies our model as a valid system of delivering Faslodex to DCIS xenografts for the purposes of endocrine manipulation and investigation.
A positive association between apoptosis and cell proliferation has been documented previously (14 , 30) and is also seen in our experiments within DCIS in day 0 specimens (i.e., breast biopsy tissue). Dysregulation of the critical balance between cell proliferation and cell death by processes that favor proliferation or impair apoptosis can result in progression of benign lesions to frankly invasive tumors (31) . However, if the balance is tipped in favor of cell death over proliferation, then tumor regression may be expected (32) .
The NSABP B-24 study randomized women with DCIS who underwent breast-conserving surgery with adjuvant radiotherapy to receive tamoxifen or placebo (3) . The study demonstrated a reduction in ipsilateral invasive cancer (but not DCIS) recurrences after the addition of tamoxifen. Unfortunately, information regarding ER status of DCIS lesions was not provided. Our data suggest that the benefit of endocrine manipulation will be limited to women with ER+ DCIS lesions. Women with ER- DCIS are unlikely to respond to AE exposure, and in these patients the side effect profile of tamoxifen treatment would make such treatment difficult to justify.
To achieve a greater response to treatment in women with ER- DCIS, a target other than the ER may need to be used. The c-erbB-2 oncogene has been shown to be present on 90–100% of comedo DCIS lesions (33) . Antibodies to this receptor molecule have been shown in vitro to induce cellular differentiation (34) and in vivo to inhibit tumor growth when radiolabeled (35) . The c-erbB-2 oncogene may, therefore, represent a potentially effective target for antigen-specific immunotherapy of breast DCIS. Studies are currently underway in our laboratory, using the described model, to investigate this possibility.
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FOOTNOTES |
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1 Supported by grants from the Tom Jones Research
Fellowship, the University of Manchester (to A. G.), and the
Association for International Cancer Research (N. J. B.); and funding
by the Cancer Research Campaign (C. S. P.). 
2 To whom requests for reprints should be
addressed, at University Department of Surgery, University Hospital of
South Manchester, Nell Lane, Manchester M20 2LR, United Kingdom. Phone:
44-161-291-3842; Fax: 44-161-291-3846; E-mail: bundredn{at}fsl.with.man.ac.uk
3 The abbreviations used are: DCIS, ductal
carcinoma in situ; AE, antiestrogen; AI, apoptotic
index; CI, confidence interval; ER, estrogen receptor; ISEL, in
situ end labeling; LI, Ki67 labeling index; PR, progesterone
receptor; NSABP, National Surgical Adjuvant Breast and Bowel Project.
Received 11/29/99. Accepted 5/24/00.
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