Trial Information

Randomized Controlled Trial of Neo-adjuvant Progesterone and Vitamin D3 in Women With LOBC and LABC - A Feasibility Study

Introduction:

- Primary Progesterone Timing of surgery during the menstrual cycle and its impact on
survival in pre-menopausal women with operable breast cancer has been extensively researched
and reinvestigated by Badwe et al in the randomized clinical trial of 'Primary Progesterone
Therapy for Operable Breast Cancer' at Tata Memorial Hospital. The underlying assumption was
that the presence of unopposed estrogen (in follicular phase) at the time surgery may be
deleterious for survival and that circulating progesterone might counteract this deleterious
effect.

The timing of surgery during the menstrual cycle and survival in breast cancer carries two
distinct postulates. First, hormonal milieu in the host can modulate metastatic potential of
breast cancer and second, events at the time of surgery can influence survival.

The trial by Badwe et al was a prospective, randomized controlled trial, in which a luteal
phase was artificially created by a depot preparation of 500mg of injectable hydroxy
progesterone caproate (Proluton). The trial design was a simple two-arm randomization
comparing standard treatment with neo-adjuvant progesterone treatment 5-14 days prior to
surgery. All patients with operable breast cancer were eligible for the protocol. A total of
1000 patients were accrued in the study which had 80% power to detect a 10% improvement over
the existing survival of 60% at 5 years. All patients were stratified for menopausal status
and tumour size. The endpoints of the study were recurrence of breast cancer and death. All
accrued patients are being regularly followed up for at least 5 years which would be the
main objective of this study. An interim analysis was carried out recently by the Data
Monitoring Committee the results of which are not yet published.

In light of the interim results of the randomized trial of primary progesterone in women
with operable breast cancer showing a significant difference in the node positive high risk
tumors (results not yet published as per the decision of the Data Monitoring and Safety
Committee), it is logical to now test the role of primary progesterone in large operable and
locally advanced breast cancer.

Progesterone hormone is known to down regulate the vascular endothelial growth factors and
epidermal growth factors which are required for successful cancer metastasis to take place.
The hormonal milieu in the host, in presence of progesterone, can thus possibly lower the
metastatic potential of breast cancer and improve survival outcome.

It has been shown by Pachmann K et al that concurrent with tumor size reduction following
primary tumor chemotherapy in breast cancer there is a massive cell release into
circulation. The impact of these released cells on outcome needs to be investigated
including possibility of altering the metastatic potential of these released cells, possibly
by altering the hormonal milieu.

- Vitamin D3 The most prominent physiological role of hormonally active form of vitamin D3,
1,25-dihydroxyvitamin D3 (1,25(OH)2D3 or calcitriol), is regulation of calcium and
phosphorous homeostasis and bone metabolism via an intracellular receptor (VDR) which is a
member of steroid thyroid hormone super-family of receptors. The VDR receptors are also
found in other tissues like breast and prostate.

The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has been
recognized for over 2 decades as a modulator of cell proliferation and differentiation
(cytotoxic and anti-proliferative) in many cell types, including breast cancer. However, it
is still unclear as to how 1,25(OH)2D3 and its synthetic analogues act within breast cancer
cells to elicit the effects on cellular proliferation and differentiation.Vitamin D3 has
been shown to decrease tumor growth in animal models and is now in Phase I clinical trials
for human cancers.

It has been found so far that 1,25(OH)2D3 has an effect on the expression of certain cell
cycle regulators and in this way can bring about G1 arrest.[9] Evidence has also emerged
that vitamin D compounds can also affect the growth-promoting pathways initiated by two
important factors involved in breast cancer cell promotion; namely the insulin-like growth
factor I (IGF-I) and oestrogen-receptor (ER) pathways.

Vitamin D compounds have also been implicated in promotion of apoptosis in breast cancer
cells and evidence suggests that 1,25(OH)2D3 and its synthetic analogues may potentiate
responsiveness of breast cancer cells to conventional cytotoxic agents. [11] Some of the
Vitamin D3 analogs have been found to enhance response to adriamycin and irradiation in
MCF-7 breast tumor cells.In combination with tamoxifen, [12] there is inhibition of breast
cancer cell growth. It also potentiates the effect of radiation therapy.Currently studies
are also ongoing evaluating Vitamin D2 as a possible alternative with lower calcaemic
effect.A phase I trial was recently reported evaluating the maximum tolerated dose and the
pharmacokinetics of oral calcitriol in conjunction with paclitaxel chemotherapy in patients
with advanced solid tumors, wherein it was found to be safe with no dose-limiting toxicity
reported in the study.

- Immunohistochemistry markers The various markers correlating with proliferation,
apoptosis, angiogenesis to be assessed are as follows MIB 1 - Women with ER positive tumors
in the luteal phase are known to have better outcome.This was because the tumors were better
differentiated and had low proliferation as demonstrated by a low MIB1 staining. On the
other hand Cooper et al found no difference in cell proliferation as measured by MIB1
staining in the two phases of the menstrual cycle.

Bcl2: One study focused on the bcl2 expression in the normal breast within the two phases of
the menstrual cycle. An inverse correlation was found between progesterone levels and bcl2
expression in apparently normal breast, bcl2 levels being higher during the follicular
phase.

Tumor Angiogenesis (CD 31 & 34) in breast cancer has been the focus in the recent years as
it is associated with poor prognosis and also presents us with opportunities to use anti
angiogenetic drugs to treat breast cancer VEGF (vascular endothelial growth factor) is a
very potent angiogenic cytokine and is known to affect overall survival and recurrence free
survival in breast cancer. It acts by stimulating tumor cell proliferation also. In a
recent study it was found that serum VEGF was lowered in the luteal phase of menstrual cycle
and had inverse relation to the serum progesterone levels. The lowering of a potential
angiogenic cytokine in the luteal phase suggests a possible decreased potential for
establishment of micrometastases during that phase.

- Objectives

1. To see the effect of primary progesterone on survival in women with high risk breast
cancer (large operable and locally advanced breast cancer)

2. To see the effect of Vitamin D3 as an anti-proliferative, cytotoxic and apoptotic agent
(negative growth regulator) by evaluation of surrogate markers of proliferation and
apoptosis.

Methodology / Treatment plan

The study drugs (Injectable Progesterone and Vitamin D3) will be tested in the neoadjuvant
setting prior to administration of each chemotherapy cycle, in a 2x2 factorial design as
below:

Neo-adjuvant D3 will be administered as Inj. Arachitol 300,000 IU/ml intramuscular, before
each chemotherapy cycle.

Neo-adjuvant Progesterone used will be administered as single IM depot injection 500mg 5
days prior to each CT cycle and surgery date.

The first incision / core biopsy will be carried out at the time of randomization. The
intervention will be administered as per assigned arm (Vitamin D3 / Proluton /Vitamin
D3+Proluton / Control). The second Core/incision biopsy will be performed before starting
NACT to evaluate the in-vivo modulation by progesterone and/or vitamin D3. Gene expression
profiles will be compared later in subsequent studies.

These patients will be assessed for response and undergo loco-regional treatment with
surgery or local radiotherapy or combined.

The study will be carried out in two phases:

Phase I: Feasibility and toxicity study (N=120 patients, 30 in each arm) Phase II: Total
accrual of 800 patients (including 120 from feasibility study) The current study is the 1st
phase of feasibility and toxicity evaluation in 120 patients.

Statistical Consideration For an expected 35% 5 year disease free survival (DFS) for the
control group, the expected 5 years DFS for the chemotherapy group will be 45%. With a
two-sided analysis, and at 80% power and 95% confidence to detect a 10% difference between
the arms, 708 patients are required at 425 events. Assuming a 10% loss to follow up, a total
of 800 patients would be accrued, with 200 patients in each arm, and an interim analysis
performed at 50% events. We expect to complete accrual of 120 patients in 2 years. The total
accrual will be possible within 5 years. A multicenter collaboration should enable the
expected accrual in five years.

Randomization and Stratification:

Randomization for chemotherapy will be done centrally from Clinical Research Secretariat and
patient will be stratified by

1. Menopausal status: Pre + Peri or Post

2. LOBC or LABC

3. Type of chemotherapy: Taxane or non-taxane

Systemic treatment:

All patients will receive CAF / CEF / TAC chemotherapy. The choice of chemotherapy schedule
will be left to the discretion of the treating oncologist. Patients who are hormone receptor
positive will be given tamoxifen or aromatase inhibitors sequentially.

End-points

Current Sub-Study:

Surrogate markers to be analyzed Clinical: Response to chemotherapy Laboratory: VEGF
(Proliferation, angiogenesis) Bcl2 (Apoptosis), TUNEL (Apoptosis),CD31 (Angiogenesis),CD34
(Angiogenesis),MiB1 (Proliferation) The tumor samples and blood collected will be stored for
any other future relevant molecular analysis (evaluation of circulating tumor cells & DNA
microarray studies).

Response Assessment:

All patients will have assessment of response at each cycle defined as CR, PR, SD and PD.
This will also be used as a surrogate end-point.

Toxicity recording:

During chemotherapy the blood counts and biochemistry will be done prior to every cycle. The
chemotherapy schedule will be postponed if the total white blood cell counts is <4000/cmm or
the absolute neutrophil count is <1500/cmm. Subsequent cycle will be given only once the
total counts normalize and the patient is clinically stable. Dosage in subsequent cycles
will be given with a 25% dose reduction of the myelosuppressive drugs or full dose with
secondary prophylaxis with growth factors.

Chemotherapy will be delayed for alteration in renal and hepatic functions and will be
restarted once they settle down to accepted values.

Serum Bilirubin: < 2.0 mg/dl SGOT/ SGPT: < 4 x upper limit of normal Serum Creatinine: < 1.5
mg/dl

Toxicity with test drugs:

Progesterone is already tested in the neo-adjuvant setting and has no toxicity noted.
Vitamin D3 has undergone phase 1 studies and in the doses prescribed has been found safe.
Published cases of vitamin D toxicity with hypercalcemia, for which the 25(OH)D
concentration and vitamin D dose are known, all involve intake of > or = 1000 microg (40000
IU)/d.

Disease Failure and Follow -Up Primary end point: First confirmation of recurrence (local,
regional or distant), second primary breast tumor and / or death. The date of treatment
failure is the date of first appearance of a suspicious lesion, later proven to be a
definite recurrence or metastasis.

All patients will be followed up at 6 monthly intervals in the first one year for a total of
5 years. After 5 years patient will follow up annually till 10 years followed by phone
contact/ postcard contact to verify long term survival till progression or death. Second
primary in the contralateral breast will be taken as progression of disease. The trial
office will be informed about the status of patients every six months. The primary endpoint
will be progression of disease and death. New cancer in distinct organ will be noted but
will not be an endpoint.

During follow up a mammography study will be done 6 months after completion of the local
treatment (surgery and or radiotherapy). Thereafter it will be done at 1.5 to 2 years
interval throughout the follow up period and at anytime it is medically indicated.

Follow up investigations will be performed based on patients' symptoms as per standard
protocol. Similarly the bone scan will be repeated if the patients' complaints warrant a
bone scan.

Analysis Disease Free Survival (DFS) will be calculated from the date of randomization to
the date of local, regional or distant relapse or death from any cause and will be censored
at the last date of follow up for the patients that are alive and disease free or have been
lost to follow up.

Overall Survival (OS) will be calculated from the date of randomization to the date of death
or censored at the date of last follow up for the patients who are alive or lost to follow
up.

DFS and OS will be evaluated using Kaplan Meier and compared by Log-rank test. The Cox
proportional hazard model will be used to assess the impact of intervention after correction
for stratification, menopausal status and age.