High Dose Estrogen Therapy for Breast Cancer

1
High Dose Estrogen Therapy for Breast Cancer

• Aruna Kommareddy, M.D.
• Fellow
• Washington University

2
Early reports

• Large doses of estrogens experimentally in
  animals
• Produced dwarfing
• Resulted in little or no breast development
• Haddow observed that large doses of estrogen
  retarded tumor growth

3
Premenopausal women advanced breast cancer

• Mainly treated with androgens and responses upto
  20 were obtained.
• Some authors also reported responses to high dose
  estrogens
• Some studies did not demonstrate any responses

4
Postmenopausal women advanced breast cancer

• In the early 1950s and 1960s most
  postmenopausal patients with advanced breast
  cancer were treated with high dose estrogen
  therapy, as other options were limited.
• Good objective responses were obtained in the
  range of 14 to 41 using different estrogens
  including DES, ethinyl estradiol, dienestrol,
  estradiol dipropionate.

5
Side effects(Retrospective analysis)JAMA July
1953
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Side effects
7
Side Effects
8
Side effects
9
Diethylstilbestrol Recommended dosages for
different categories of breast cancer
patientsCooperative breast cancer group

• A total of 523 postmenopausal women with
  metastatic breast cancer were entered in a
  randomized double blind study of 4 doses of DES
  1.5mg, 15mg, 150mg, 1500mg/d
• 16 institutions participated.
• Patients with no prior hormonal therapy and
  measurable disease were admitted
• Objective response If more than 50 of disease
  sites responded and others stable with no new
  lesions.
• Monitored by serial x rays and clinical
  measurements. 
• 1977 JAMA may 9, 1977-Vol 237, No 19

10
Randomization

• Prior to randomization each patient was
  classified by postmenopausal age 1 year, 1-5
  years, 5-10 years, more than 10 years.
• By dominant metastatic site.
• The patient populations for the 4 dosages were
  not significantly different
• Therapy was continued for a minimum of 12 weeks
  unless compelling progression of disease
  intervened.

11
Results

• Higher doses produced higher regression (plt .05)
  21 for 1500 mg dosage
• 17 for 150 mg
• 15 for 15 mg
• 10 for 1.5 mg
• Duration of regression were similar regardless
  of dose
• Median duration of remission for responders was
  around 13 months
• No regressions were obtained in women who were
  less than 1 year postmenopausal no matter what
  the dosage.

12
Results

• Survival significantly improved for responders
• No withdrawal regressions were seen at any dosage
  in this group of patients 
• 15 had rapidly progressive disease
• Toxicities increased with increasing dosage.
• Included anorexia, nausea, vomiting mastalgia,
  vaginal bleeding, CHF, hypercalcemia

13
Randomized clinical trial of DES versus Tamoxifen
in postmenopausal women with advanced Breast
cancer.

• The trial involved 143 evaluable patients
• Enrolled between Mar 77-Feb 80
• Had to be 5 or more years since LMP
• PS ECOG 3 or better
• Estrogen receptor analysis was not mandatory and
  was determined by a dextran coated charcoal
  technique
• Was available in 12 patients in the DES arm and
  11 patients in the Tamoxifen arm
• N Engl J Med. 1981 Jan 1304(1)16-21

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Schema
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Patient Characteristics
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DES Vs Tam

• The regression rates (CRPR) were higher in
  patients receiving DES (41 per cent) than in
  those receiving tamoxifen (33 per cent), but not
  significantly (P 0.37).
• Analysis of the time until treatment failure for
  the two treatment groups showed no significant
  difference (medians DES, 142 days tamoxifen,
  171 days).
• Toxicity was greater in patients receiving DES
• 9/74 patients (12) discontinued therapy solely
  because of adverse reactions.

17
DES VS Tam

• Tamoxifen became the standard of care
  subsequently because of equivalent efficacy and
  less toxicity

18
Updated Analysis 1999Breast Cancer Res treat

• All patients were followed until death or for a
  minimum of 14.1 years on DES arm and 16.7 years
  on the TAM arm.
• The overall ORR for DES was 42 (95 confidence
  interval (CI) 3154).
• The overall ORR in patients receiving TAM was 33
  (95 CI 2246).
• The median response time was 11.8 months for DES
  and 9.9 months for TAM.

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Updated Analysis 1999

• Of the 143 eligible patients, 139 had a
  documented progression.
• The median time-to-disease progression was 6.9
  months (95 CI 4.710.1 months) in the patients
  on DES and 5.9 months (95 CI 48.3 months) in
  the patients on TAM
• The progression hazard ratio (TAM/DES) was 1.07
  (95 CI 0.771.49).

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DES Vs TAM

• Of the 143 eligible patients, 137 died.
• On the DES arm, the median survival was 3.0 years
  (95 CI2.64.7 years) and the 5-year survival
  was 35 (95CI 2648).
• On the TAM arm, the median survival was 2.4 years
  (95 CI 1.73.4 years) and the 5-year survival
  was 16 (95 CI 927).
• Survival among women on DES was significantly
  increased over that of women on the TAM arm
• Multivariate Cox regression analysis indicated
  PFS was significantly increased for women with
  soft tissue dominant disease and no history of
  prior chemotherapy.

21
Results
22
DES Vs TAM

• 18 patients who had achieved a response on DES
  entered a withdrawal phase and 5 (28) achieved
  an OR.
• The median time to treatment failure was 3.2
  months.
• 16 patients who progressed on the DES withdrawal
  phase were subsequently treated with TAM and 5
  achieved an objective response (2 CR, 3 PR).
• The median time to treatment failure was 7.0
  months.
• Two patients were mistakenly given TAM
  immediately following progression after a PR on
  DES and both achieved a response with TAM (1 CR,
  1 PR).

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Estradiol
24
DES
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High Dose estrogen and breast cancer

• The activity of high dose estrogen in breast
  cancer is paradoxical because low doses of
  estrogen in the form of hormone replacement
  therapy have been positively associated with a
  risk of developing breast cancer, and breast
  cancer in postmenopausal women is usually managed
  by extreme estrogen deprivation with third
  generation aromatase inhibitors

26
In Vitro Studies

• In vitro studies have provided insight into
  possible mechanisms of action of high doses of
  estrogen
• Indicate that long-term estrogen deprivation may
  increase sensitivity to the therapeutic effects
  of pharmacologic doses of estrogen.
• This is of considerable interest because the
  shift towards the use of aromatase inhibitors for
  the adjuvant treatment of breast cancer means
  that the majority of postmenopausal women with
  advanced disease will have already been treated
  with extreme estrogen deprivation..

27
LTED and MCF-7 cells

• Long term estrogen deprived cells (LTED) are
  derived from MCF- 7 breast cancer cells through
  growth under low estrogen conditions and mimic
  the hormonal milieu of breast cancer in
  postmenopausal women receiving an aromatase
  inhibitor
• Song et al conducted experiments on LTED cells to
  investigate the mechanism by which high dose
  estradiol causes breast tumor regression in
  postmenopausal women.
• They assessed the effect of varying
  concentrations of estradiol or vehicle (ethanol)
  on the growth of LTED and MCF-7 cells.

28
Results

• Treatment of the LTED cells with estradiol at 0.1
  nM and 10 nM resulted in 60 lower cell numbers
  compared with vehicle treatment. (Plt. 001)
• Estradiol at a concentration of 10 nM did not
  produce a further reduction in cell number
  compared to 0.1 nM estradiol.
• Estradiol increased MCF-7 cell growth in a
  dose-dependent fashion.
• LTED cells showed a sevenfold increase in
  apoptosis (plt. 001) relative to vehicle-treated
  cells at an estradiol concentration of 0.1 nM,
  while apoptosis of MCF-7 cells was diminished in
  the presence of estradiol

29
Effect of 17 estradiol (E2)on numbers of LTED
and MCF-7 cells.

• Cells were treated with E2 or with vehicle
  (ethanol) for 4 days.
• 1E-05 is a designation for 0.00001 nM E2.
• Points represent the means of cell count for one
  representative experiment run in triplicate.
• Error bars represent 95 confidence intervals of
  the means.

30
Effect of E2 on annexin V membrane binding

• Cells were treated with vehicle or with 0.1 nM E2
  for 12 hours.
• Apoptosis was observed by annexin V-FITC binding
  to the cell membrane.

31
Concentration dependence of E2's effect on
apoptosis in LTED and MCF-7 cells

• Cells were treated with the indicated
  concentrations of E2 for 3 days
• DNA fragmentation of apoptosis was assayed by the
  ELISA method
• The data represent the fold increase of the means
  over the control (i.e., vehicle-treated cells)
  for an experiment run in triplicate.

32
Analysis of DNA fragmentation by agarose gel
electrophoresis

• LTED cells were treated with vehicle or 0.1 nM E2
  for 3 days.
• Genomic DNA was extracted and analyzed on a 0.5
  agarose gel.
• Lane 1 was loaded with 1-kilobase DNA ladder as a
  molecular weight control.
• DNA samples loaded on gel are from
  vehicle-treated LTED cells (lane 2) and
  E2-treated LTED cells (lane 3).

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Effect of E2 on the morphology of LTEDand MCF-7
cells

• Cells were treated with vehicle or with 0.1 nM E2
  for 3 days.
• Photographs were taken with a phase-contrast
  microscope every day for LTED cells and on day 3
  for MCF-7 cells.
• Apoptotic changes presence of phase-bright cell
  bodies, shrunken cytoplasm, and an increased
  number of floating cells.

34
Estrogen receptor involvement in 17 -estradiol
(E2) action on apoptosis

• LTED cells were pretreated with different
  concentrations of the ICI 182780 (ICI) for 30
  minutes and then treated with vehicle (open bars)
  or with 0.1 nM E2 (solid bars) for 3 days.
• DNA fragmentation, a measure of apoptosis, was
  assessed by ELISA.
• The data represent the means of the fold
  induction over the vehicle-treated control for
  one of the experiments run in triplicate.

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Fas/Fas L pathway

• Song et al hypothesized that Fas/Fas L pathway
  could be involved in the apoptotic process.
• Fas is one of the most important genes involved
  in apoptosis.
• It is a membrane receptor and cross linking of
  Fas through the binding of its natural ligand Fas
  L or an agonistic anti-Fas antibody induces cell
  death by apoptosis.
• MCF-7 cells have FasL but no detectable Fas, LTED
  cell express both Fas and FasL and increased
  amounts of Fas protein appear in response to
  long-term estrogen deprivation.

36
Fas/Fas L

• Compared to treatment with vehicle alone,
  estradiol treatment increased FasL protein
  expression in LTED cells and MCF-7 cells.
• Expression of Fas protein was not affected in
  either cell lines by estradiol treatment.
• This demonstrates that increased Fas protein in
  LTED cells might provide sufficient receptor for
  estradiol induced increased FasL to function,
  leading to apoptosis

37
Western blot analysis of Fas and Fas ligand
(FasL) expression

• LTED, MCF-7, and MCF-10A cells
• (A) were cultured in Iscove's modified Eagle's
  medium containing 1 dextran-coated
  charcoal-stripped fetal bovine serum.
• LTED cells (B) were treated with vehicle (i.e.,
  control) or with 0.1 nM 17 -estradiol (E2) for
  the times indicated.
• Cells were then extracted with Trizol reagent,
  and the expression of Fas and FasL was measured
  by western blot analysis.
• The molecular masses of Fas (46 kilodaltons
  kDa) and of FasL (30 kDa) are indicated.
• Lower panels show quantitative densitometric
  scanning results from three independent
  experiments.

38
Anti Fas Antibody

• Subsequent experiments demonstrated that
  treatment of cells with the agonistic anti-Fas
  antibody or with estradiol at 0.1 nM stimulated
  LTED cell apoptosis to levels twofold and
  threefold over those in vehicle-treated cells,
  respectively.
• Furthermore, a combination of anti-Fas antibody
  with estradiol had an additive effect on
  apoptosis of LTED cells.
• MCF-7 cells were resistant to the effects of the
  antibody on apoptosis, which was consistent with
  their lack of Fas protein expression.
• All the above in vitro studies provide possible
  explanations for the activity of high doses of
  estrogen in postmenopausal women

39
Effects of activating anti-Fas antibody and the
pan-caspase inhibitor Z-VAD on 17 -estradiol
(E2)-induced apoptosis

• A) Effect of activating anti-Fas antibody on
  E2-induced apoptosis of LTED cells.
• LTED cells (left panel) were treated with 0.5
  µg/mL monoclonal anti-Fas (anti-Fas mAb) or with
  0.5 µg/mL isotype-matched control monoclonal
  antibody (control mAb) in the presence or absence
  of 0.1 nM E2.
• MCF-10A and MCF-7 cells (right panel), treated
  with control mAb or anti-Fas mAb, were used as
  positive and negative controls for LTED cells,
  respectively.
• B) LTED cells were pretreated with various
  concentrations of Z-VAD for 30 minutes and then
  treated with vehicle (open bars) or 0.1 nM E2
  (solid bars) for 3 days.
• In both sets of experiments, DNA fragmentation, a
  measure of apoptosis, was assessed by ELISA.
• The data represent the increased apoptosis as
  fold of the vehicle-treated control from an
  experiment performed in triplicate.

40
Role Of IGF

• High plasma levels of IGF-I have been reported to
  be a risk factor for development of breast cancer
  in pre-menopausal women.
• Anti-tumor effects were demonstrated in animal
  models using antibodies to inhibit IGF-I.
• Helle et al measured the plasma levels of IGF I,
  IGF II and free IGF I in patients with metastatic
  breast cancer receiving treatment with DES,
  before and after treatment and reported
  significant reductions in the levels of IGF-I,
  IGF II and free IGF-I post treatment.
•  

41
High-dose estrogen Rx in postmenopausal breast
cancer patientsheavily exposed to endocrine
therapy

• 32 patients were enrolled in the protocol
• Median age was 68 years (range 4587 years).
• Each patient should have documented disease
  progression at the time of enrolment.
• Patients should have received and become
  resistant to previous endocrine therapy given at
  relapse
• Previous chemotherapy (adjuvant and/or for
  advanced disease) was allowed.
• Breast Cancer Research and Treatment 67 111116,
  2001.

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Patient characteristics
43
Characteristics
44
Results

• 19 patients stopped therapy secondary to side
  effects
• 10/32 patients, (31) obtained an objective
  response to therapy.
• 8/10 patients who obtained an objective response
  had their response recorded on at least two
  different occasions 4 weeks apart.
• Median duration of response was 50 weeks (range
  30124 weeks), Notably, five patients had an
  objective response lasting for more than 52
  weeks.
• In addition to those obtaining tumor regression,
  two patients obtained stabilization of their
  disease for 6 months, suggesting a total of 12
  (38) to benefit from therapy.
• Median time to progression (for all patients) was
  18 weeks, while the median time to failure was
  16.5 weeks.

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Results
46
Conclusions

• High Dose estrogen therapy does have a role in
  the management of advanced breast cancer.
• In the second line endocrine therapy setting in
  patients having received tamoxifen the RR
  observed for anastrazole, exemestane and
  letrozole are 10, 15 and 24 respectively
• High dose estrogen has shown about 31 RR in
  heavily pretreated patients in a small study
• Exact dose is not known
• Patients with extreme estrogen deprivation may
  respond to low doses
• Larger studies are needed