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Vol. 12, 34 – 41, January 2003
Cancer Epidemiology, Biomarkers & Prevention
Fenretinide Breast Cancer Prevention Trial: Drug and Retinol Plasma
Levels in Relation to Age and Disease Outcome1
Franca Formelli,2 Tiziana Camerini, Elena Cavadini,
Valentina Appierto, Maria Grazia Villani, Alberto Costa,
Giuseppe De Palo, Maria Gaetana Di Mauro, and
Umberto Veronesi
Istituto Nazionale Tumori, 20133 Milan, [F.F., T.C., E.C., V.A., M.G.V.,
G.D.P., M.G.D.M.]; S. Maugeri Foundation, Pavia [A.C.]; and Istituto Europeo
di Oncologia, 20133 Milan [U.V.], Italy
Abstract
Objectives: To assess, in women participating in a breast
cancer prevention trial on fenretinide (4-HPR), the
relationship of drug and retinol levels with the risk of
second breast malignancy, taking into account age and
menopausal status.
Methods: In a multicenter prevention trial, women
with early breast cancer were randomly assigned to
receive no treatment or 200 mg of 4-HPR/day for 5 years.
Blood was collected at baseline and on a yearly basis
during intervention from women recruited at the Istituto
Tumori (Milan, Italy; 818 and 756 in the 4-HPR and
control arm, respectively, who accounted for 53% of the
participants in the trial). The plasma concentrations of
4-HPR, its main metabolite N-(4-methoxyphenyl)
retinamide, and retinol were assayed by highperformance liquid chromatography. Three age ranges
(<45, 46 –55, and >56 years), menopausal status at
baseline, and disease outcome at a median follow-up of 97
months were taken into account in the analysis.
Results: Baseline retinol levels were significantly
lower (P < 0.05) in subjects < 45 years than in older
subjects, and among subjects in the age range 46 –55
years, they were significantly higher (P < 0.001) in those
in postmenopause than in those in premenopause.
Baseline retinol levels were not related to the risk
of a second breast malignancy. 4-HPR and N-(4methoxyphenyl)retinamide levels were not affected by
menopausal status. They slightly, but significantly (P <
0.05), increased with age (>46 years versus <45 years)
but only in disease-free subjects. Among subjects < 45
years, they were slightly, but significantly (P < 0.05),
higher in those subjects in which breast cancer recurred.
4-HPR treatment caused a retinol level reduction,
Received 6/17/02; revised 9/18/02; accepted 10/10/02.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by NIH/National Cancer Institute Grants CA38567 and CA72286
and the Associazione Italiana per la Ricerca sul Cancro.
2
To whom requests for reprints should be addressed, at Istituto Nazionale
Tumori, Via Venezian 1, 20133 Milan, Italy. Phone: 39-02-23902706; Fax:
39-02-23902692; E-mail: [email protected].
which was strongly (r > 0.71; P < 0.001) related to
pretreatment retinol levels.
Conclusions: Retinol plasma levels increased with age
and after menopause and were not related to breast
cancer recurrence. 4-HPR levels were lower in subjects <
45 years than in older subjects. The inverse relationship
between drug plasma levels and 4-HPR preventive effects
observed in young women suggests a role for 4-HPR
plasma sequestration in 4-HPR biological activity.
Introduction
In March 1987, a multicenter randomized trial was started to
assess the efficacy of the synthetic retinoid 4-HPR3 as a breast
cancer preventive agent (1). The objective of the trial was to
evaluate the efficacy of a 5-year intervention with 4-HPR
versus no treatment in reducing the incidence of contralateral
breast cancer or ipsilateral breast cancer in women operated on
for early breast cancer. A different effect of 4-HPR was found
depending on menopausal status, with a possible beneficial
effect in premenopausal women and a reverse trend in postmenopausal women.
Similarly, studies on subgroups of women participating in
the prevention trial had shown that 4-HPR affected circulating
insulin-like growth factor-1 differently depending on age, with
a greater reduction in women ⱕ 50 years (mostly in premenopause) than in older women (2, 3). All together, these findings
suggest an interaction between 4-HPR effects and menopausal
status or age.
Information on 4-HPR pharmacokinetics and pharmacodynamics was obtained during a Phase I study, which was run
to identify the dose to be administered in the breast cancer
prevention study (4, 5). We demonstrated that 4-HPR, administered at 200 mg/day, resulted in average 4-HPR and 4-MPR
plasma levels of ⬃1 ␮M, which remained constant over the
5-year treatment period (4) and caused a 70% decrease in
plasma retinol levels (4, 5). In these studies, because of the few
patients investigated, it was not possible to assess the potential
influence of age or menopausal status on 4-HPR pharmacokinetics. Now with the availability of measurements of drug and
retinol levels in plasma samples collected at baseline and during
intervention from 1574 women (818 and 756 in the 4-HPR and
control arm, respectively) participating in the Phase III prevention trial, it seemed appropriate to evaluate the association
between drug plasma levels and disease outcome, taking into
account the age and menopausal status of the patients. Moreover, because few and contradictory results (6 –10) have been
reported on the potential association between vitamin A (or
retinol) and breast cancer, we assessed whether prerandomiza-
3
The abbreviations used are: 4-HPR, fenretinide; 4-MPR, N-(L-methoxyphenyl)retinamide; BMI, body mass index; RBP, retinol binding protein.
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Cancer Epidemiology, Biomarkers & Prevention
Table 1
Main subject characteristics
Fenretinide (n ⫽ 818)
Age (years)
ⱕ45
46–55
ⱖ56
Menopausal status
Premenopausal
Postmenopausal
Tumor stagea
pT1
pT2
pT1–T2
pTxb
pTisc
Control (n ⫽ 756)
n
%
n
%
184
380
254
22.5
46.5
31.0
184
343
229
24.3
45.4
30.3
392
426
47.9
52.1
392
364
51.8
48.2
586
173
13
36
10
71.6
21.2
1.6
4.4
1.2
577
138
10
26
5
76.3
18.3
1.3
3.4
0.7
a
International Union Against Cancer tumor-node-metastasis classification (Ed. 4,
Berlin, Germany, 1987).
b
pTX, primary tumor cannot be assessed.
c
pTis, carcinoma in situ.
tion or on-study retinol levels were predictive of breast cancer
risk and recurrence.
Patients and Methods
Sample Population and Treatment. The sample population
of the study consisted of 1574 women (818 in the 4-HPR arm
and 756 in the control arm, see Table 1) participating in a
multicenter 4-HPR breast tumor prevention trial (1). The
women were those followed at the Istituto Nazionale Tumori,
Milan, Italy, and they accounted for 53% of the participants in
the trial. The median follow-up duration for clinical response is
the same as the prevention trial, i.e., 97 months. Blood samples
from 83 women (blood donors: age range 28 – 67 years), with
no evidence of a breast tumor, were also collected and analyzed
for comparison of retinol levels.
The design and all study features of the prevention trial
have been described previously (1, 11). The study received
Institutional Review Board approval, and all subjects signed a
written informed consent form. Eligible subjects were women
30 –70 years old who had been operated on previously for stage
I breast cancer (T1-T2 NO) or ductal carcinoma in situ and
received no adjuvant systemic therapy. Women were assigned
randomly to receive either no treatment or 4-HPR (a gift from
R.W. Johnson Pharmaceutical Research Institute, Springhouse,
PA) given p.o. at the dose of 200 mg/day for 5 years. Patients
were advised to take the daily dose (two capsules of 100 mg
each) after dinner. The occurrence of contralateral breast cancer
or ipsilateral breast cancer was the primary end point of the
study to evaluate drug efficacy. The occurrence of distant
metastases (including regional relapse) and a second primary
cancer in organs other than the breast was also recorded. Adverse events or toxicities were assessed as described previously
(1, 11, 12).
Sample Collection and Analytical Procedure. Subjects allocated to the 4-HPR arm had plasma samples collected before
randomization (at baseline) and periodically during follow-up
visits (at least every 12 months) during the 5 years of study.
Subjects allocated to the control arm had plasma collected at
baseline and after 5 years. For each sample, the interval from
randomization (in months) was reported. For samples of the
4-HPR arm, the interval (in h) from the last dose and the
number of capsules taken (one or two) were also reported.
Blood was collected in heparinized tubes and wrapped in
aluminum foil; all procedures were performed in the dark. After
centrifugation, separated plasma was kept frozen at ⫺20°C
until analysis, never ⬎3 weeks. Aliquots of 0.2 ml of plasma
were added to 0.4 ml of CH3CN containing butylated hydroxytoluene (125 ␮g/ml; Sigma, St. Louis, MO) as antioxidant, and
the concentrations of 4-HPR, 4-MPR, and retinol were detected
by high-performance liquid chromatography as described previously (4). The reference standards 4-HPR (molecular weight,
Mr 391,000) and 4-MPR (molecular weight, Mr 405,000) were
supplied by the R.W. Johnson Pharmaceutical Research Institute; retinol, used as reference standard, was obtained from
Sigma.
Statistical Analysis. Menopausal status (before or after menopause), defined as the absence of menstruation for ⱖ1 year (1),
and age at baseline were considered. Preliminary studies were
performed on the relationship of retinol, 4-HPR, and 4-MPR
with age by regression analysis in subjects who did not develop
any evident disease and who developed contralateral and ispilateral breast cancer (i.e., the disease conditions with the highest number of subjects). The regression of retinol on age was
significant. In contrast, the regression of 4-HPR and 4-MPR
was not significant even when subjects were divided according
to menopausal status. As 4-HPR and 4-MPR were not related
with age, subjects were divided into age ranges. Three age
ranges were chosen: (a) ⱕ45; (b) 46 –55; and (c) ⱖ56 years.
The ranges ⱕ45 and ⱖ56 years were chosen because they
included mostly pre and postmenopausal women, respectively.
The range 46 –55 years was chosen because it included both pre
and postmenopausal women. As regards the regression analysis
between retinol and age, when it was performed within each age
range, it was significant only in the age range 46 –55, which
included pre and postmenopausal women. Therefore, in all of
the analyses, plasma levels were subdivided according to the
three age ranges, also taking into account menopausal status.
The levels of 4-HPR, 4-MPR, and retinol were expressed as the
mean ⫾ SD. In all analyses, the values were log transformed to
approximate a Gaussian distribution. In all statistical analyses,
groups with one to three subjects were not considered for
comparison, but their means were reported in tables for descriptive purposes.
Retinol levels at baseline were subdivided according to the
three age groups, and menopausal status and the differences
between the means were evaluated by Student’s t test (Table 2).
The baseline retinol levels were then subdivided according to
subsequent disease outcome, and the comparison of each mean
versus that of subjects with no evident disease was carried out
according to Dunnet’s t test (Fig. 1). The stability in time of
retinol levels in control subjects was evaluated by analyzing the
differences between the values at the beginning and end of the
intervention period by Student’s paired t test in subjects who
had blood drawn at both intervals (Table 3).
For 4-HPR-treated subjects, to analyze uniform time intervals during the 5-year intervention period, the following time
ranges were considered: (a) 7–18 months (1 year); (b) 19 –30
months (2 years); (c) 31– 42 months (3 years); (d) 43–54
months (4 years); and (e) 55– 66 months (5 years). The stability
in time of 4-HPR, 4-MPR, and retinol levels was checked by:
(a) regression analysis in all of the subjects with at least three
measurements at different intervals from the beginning of treatment; and (b) Student’s paired t test in subjects who had blood
drawn at 1 and 5 years (Table 4). Dunnet’s t test was carried out
for comparison of the means of 4-HPR and 4-MPR levels in
subjects with different disease outcome with those of subjects
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Fenretinide Plasma Levels and Second Breast Malignancy
Table 2 Retinol plasma levels (means ⫾ SD in ng/ml) at baseline in subjects participating in the 4-HPR breast cancer prevention trial and in female healthy subjects
4-HPR prevention trial
Age (years)
ⱕ45
46–55
ⱖ56
Menopausal
statusa
Pre
Post
Pre
Post
Pre
Post
Female healthy subjects
Group
(n)
4-HPR
(n)
CTRb
(n)
(181)
(3)
(211)
(169)
541 ⫾ 130.4
555 ⫾ 25.1
562 ⫾ 123.0c
634 ⫾ 132.5d
541 ⫾ 93.50
(10)
(8)
574 ⫾ 91.9
612 ⫾ 118.4
617 ⫾ 151.2
521 ⫾ 115.9
565 ⫾ 101.3
555 ⫾ 134.0c
620 ⫾ 157.7d
545 ⫾ 122.3
612 ⫾ 144.9
(53)
(254)
(181)
(3)
(209)
(134)
(2)
(227)
(12)
642 ⫾ 199.6
a
Menopausal status at baseline: pre, premenopausal; post, postmenopausal.
b
CTR, controls.
c
P ⱕ 0.05 versus subjects aged ⱕ45 years.
d
P ⬍ 0.001 versus the same age subjects.
of the same age with no evident disease (Table 5). For comparison of the means of the different age groups within each
type of disease condition, the Student’s t test on orthogonal
contrasts was carried out according to the following criteria: (a)
age group ⱕ 45 years versus age group 46 –55 years plus age
group ⱖ56 years; and (b) age group 46 –55 years versus age
group ⱖ 56 years (Table 5).
Correlation analysis was performed to study the relationship between each pair of variables considered for 4-HPRtreated patients in each age group. The variables considered
were: (a) 4-HPR; (b) 4-MPR; (c) retinol during treatment; (d)
retinol at baseline; (e) retinol difference (i.e., retinol at baseline
minus retinol during treatment); (f) body surface area (m2); and
(g) BMI (i.e., weight/height2 expressed in kilograms and
meters, respectively).
All of the analyses were performed using SAS software
(13). Probability values reported are two sided. Values of P ⱕ
0.05 were considered significant.
Results
Subject Characteristics and Pretreatment Plasma Retinol
Levels. The main characteristics of the subjects, whose blood
was collected during the 4-HPR breast cancer prevention trial,
are described in Table 1. The two treatment arms were well
balanced and comparable with the whole study cohort (1).
Pretreatment retinol levels of subjects randomized in the
4-HPR and control arms are reported in Table 2, and they are
compared with those of healthy female subjects. Only six
subjects aged ⱕ45 years were in postmenopause in the 4-HPR
(n ⫽ 3) and control (n ⫽ 3) arms, and only two subjects aged
ⱖ56 years were in premenopause in the control arm. A comparison between pre and postmenopause was assessed only in
the age group 46 –55 years. In this age group, postmenopausal
subjects had significantly higher retinol levels (P ⱕ 0.001) than
premenopausal subjects. Among premenopausal subjects, retinol levels were lower (P ⱕ 0.05) in those aged ⱕ45 years than
in those aged 46 –55 years. There were no significant differences in retinol levels between the 4-HPR and control arms.
The average retinol levels of subjects participating in the prevention trial were not statistically different from those of
healthy female subjects.
To assess the value of retinol levels in predicting disease
outcome and toxicity, the baseline retinol levels of subjects in
the 4-HPR and control arm were subdivided according to subsequent disease conditions or toxicity and compared with the
levels of subjects with no evident disease (Fig. 1). In the 4-HPR
arm, subjects aged ⱕ45 years who experienced toxicity during
4-HPR treatment had initial retinol levels significantly lower
(P ⱕ 0.05) than subjects of the same age with no evident
disease. In the control arm, baseline retinol levels of the different groups did not differ from those of subjects with no
evident disease, except for lower average retinol levels in
women in the age group ⱖ56 years, who subsequently developed metastasis.
Retinol Plasma Levels of Subjects in the Control Arm during the 5-year Study. The stability of retinol levels in control
subjects during the 5-year study was assessed by comparing
retinol levels in subjects who had plasma samples drawn at
baseline and at 5 years (Table 3). There were no changes from
0 to 5 years except for a significant increase in subjects aged
46 –55 years who were in premenopause at baseline and had no
evident disease.
4-HPR, 4-MPR, and Retinol Plasma Levels during the
5-year Study. For analysis of samples during 4-HPR intervention, the following criteria of selection were used to obtain
consistent data: (a) last drug intake dose, 200 mg as one and not
as two separate 100-mg daily doses; (b) blood sampling at
10 –18 h from drug intake, i.e., between peak and through
concentrations at steady state (4); and (c) samples with 4-HPR
levels ⱖ 150 ng/ml, corresponding on the basis of previous
kinetics study (14), to the lower 95% tolerance limit for drug
concentration at 18 h. After the selection criteria, 2348 samples
from 537 subjects in the 4-HPR arm were assessable for analysis of drug and retinol levels during the 5-year intervention
period.
The eventual stability of 4-HPR, 4-MPR, and retinol levels
during the intervention period was first assessed. To this aim,
regression analysis was performed on 4-HPR, 4-MPR, and
retinol levels for each subject with samples drawn at least at
three different intervals during treatment. Among the 298 subjects analyzed, the regressions were significant only in very few
cases (ⱕ6% for 4-HPR and 4-MPR and 13% for retinol). The
stability of drug and retinol levels was further analyzed by the
paired t test in plasma levels of women who had plasma
samples drawn at 1 and 5 years (Table 4). No changes in 4-HPR
and 4-MPR plasma levels from 1 to 5 years were found. Retinol
levels after the 5-year treatment were slightly lower than the
levels at 1 year, and the differences were significant in the
groups with no evident disease (P ⱕ 0.05 and 0.001). In the age
group 46 –55 years, no differences were found between the
means of pre and postmenopausal women at 1 or 5 years.
Therefore, all of the following analyses on subjects in the
4-HPR arm were performed, taking into account only the age
groups and disease outcome.
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Cancer Epidemiology, Biomarkers & Prevention
Fig. 1. Plasma retinol levels (means ⫾ SD) at
baseline according to subsequent disease outcome and toxicity (data referring to fewer than
three subjects are not reported). Empty columns, subjects in premenopause; gray columns,
subjects in postmenopause. The number of subjects belonging to each group is reported inside
the columns. CTR, controls; Cont. or ipsi. ca.,
contralateral or ipsilateral breast cancer; Met.,
metastasis; 2nd primary ca., second primary
cancer; Tox., toxicity or abnormal laboratory
tests. *, P ⱕ 0.05 versus same age subjects with
no evident disease. Dunnet’s t test.
Comparison of 4-HPR, 4-MPR, and Retinol Plasma Levels
after 1 Year of Treatment. After the observation of drug and
retinol stability during the 5-year treatment, comparison of
mean levels for the different age groups according to disease
outcomes and toxicity was performed at 1 year, i.e., when more
samples were available. The mean levels of 4-HPR, 4-MPR,
and retinol of subjects whose plasma was drawn after 1 year of
treatment and the mean values of body surface area and BMI at
baseline are reported in Table 5. The proportion of subjects,
reported in Table 5, initially operated on for carcinoma in situ
pT1 and pT2 who subsequently developed contralateral or
ipsilateral breast cancer and metastasis was similar to that of
subjects of the same age with no evident disease (data not
shown).
Comparison of plasma levels was first assessed among age
groups. In subjects with no evident disease, the 4-HPR and
4-MPR levels of the groups 46 –55 and ⱖ56 years, which were
not different from one another, were significantly higher (P ⱕ
0.05) than those of the group ⱕ45 years, despite concomitant
larger body surface areas and BMI (P ⱕ 0.005). No difference
was found among age groups in the other disease conditions.
The plasma levels of subjects with the different disease
conditions were then compared with those of subjects with no
evident disease. In subjects ⱕ 45 years, with contralateral or
ipsilateral breast cancer, the means of 4-HPR and 4-MPR levels
were higher (P ⱕ 0.05), and the means of retinol levels were
lower (P ⱕ 0.05) than those of subjects of the same age with no
evident disease. In subjects of all age groups who developed
metastasis, average 4-HPR and 4-MPR levels were higher than
in subjects with no evident disease, although the differences
were not statistically significant. In these subjects, the SD of
4-MPR levels was also higher. This was attributable to many
subjects with abnormally high 4-MPR levels, i.e., ⱖ900 ng/ml.
Among subjects who developed metastasis, those ⱖ56 years
had a larger body surface area and higher BMI than subjects
with no evident disease of the same age. The few subjects who
experienced toxicity had smaller body surface areas (mean
values 1.41 and 1.54 m2) than subjects in the other groups (the
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Fenretinide Plasma Levels and Second Breast Malignancy
Table 3
Plasma retinol levels (means ⫾ SD in ng/ml) in control patients at baseline and after 5 years
Subjects with
Age (years)
Menopausal
statusa
Year
Contralateral or
ipsilateral breast cancer
(n)
No evident disease
(n)
ⱕ45
Pre
46–55
Pre
0
5
0
5
0
5
0
5
Post
ⱖ56
a
b
Post
(63)
(76)
(49)
(82)
517 ⫾ 106.5
528 ⫾ 133.8
547 ⫾ 126.6
597 ⫾ 122.8b
597 ⫾ 154.4
593 ⫾ 131.6
611 ⫾ 154.8
603 ⫾ 151.4
(9)
(12)
(5)
(6)
493 ⫾ 88.3
533 ⫾ 118.0
522 ⫾ 83.5
515 ⫾ 114.3
549 ⫾ 81.6
547 ⫾ 161.6
546 ⫾ 130.9
593 ⫾ 61.9
Metastasis
Second primary cancer
(n)
(7)
(3)
(9)
(n)
518 ⫾ 180.9
627 ⫾ 215.7
616 ⫾ 229.0
428 ⫾ 198.4
621 ⫾ 170.0
594 ⫾ 99.9
(4)
684 ⫾ 170.8
638 ⫾ 64.1
Menopausal status at baseline: pre, premenopausal; post, postmenopausal.
P ⱕ 0.01 versus baseline (year 0) by Student’s t paired test.
Table 4
4-HPR, 4-MPR, and retinol plasma levels (means ⫾ SD in ng/ml) after 1- and 5-year 4-HPR treatments
Subjects with
Treatment
(year)
Age (year)
ⱕ45
4-HPR
4-MPR
Retinol
46–55
4-HPR
4-MPR
Retinol
ⱖ56
4-HPR
4-MPR
Retinol
a
b
1
5
1
5
1
5
1
5
1
5
1
5
1
5
1
5
1
5
No evident disease
(n)
Premenopause
(42)
385 ⫾ 118.6
354 ⫾ 115.3
372 ⫾ 156.3
348 ⫾ 121.2
156 ⫾ 85.3
136 ⫾ 67.3a
446 ⫾ 137.2
452 ⫾ 149.4
437 ⫾ 209.0
456 ⫾ 212.6
164 ⫾ 84.7
144 ⫾ 79.8a
(35)
(n)
(24)
(26)
Contralateral or ipsilateral breast cancer
Postmenopause
459 ⫾ 146.6
414 ⫾ 145.3
441 ⫾ 178.2
433 ⫾ 209.7
174 ⫾ 88.9
141 ⫾ 65.5a
435 ⫾ 137.6
445 ⫾ 154.3
460 ⫾ 277.2
480 ⫾ 273.5
171 ⫾ 78.6
136 ⫾ 88.0b
(n)
Premenopause
(4)
554 ⫾ 241.8
489 ⫾ 154.5
463 ⫾ 110.1
540 ⫾ 161.8
124 ⫾ 100.5
73 ⫾ 41.0
425 ⫾ 65.1
348 ⫾ 102.9
425 ⫾ 95.7
407 ⫾ 163.2
177 ⫾ 18.7
165 ⫾ 60.1
(5)
(n)
Postmenopause
(4)
473 ⫾ 67.8
424 ⫾ 42.7
415 ⫾ 86.5
396 ⫾ 86.4
211 ⫾ 132.0
120 ⫾ 61.9
P ⱕ 0.05.
P ⱕ 0.001 versus 1-year levels by Student’s paired t test.
lowest mean value was 1.59 m2 for subjects ⱕ 45 years with
no evident disease), suggesting a proportionally higher drug
intake.
The correlations between each pair of variables reported in
Table 5 were also assessed. Retinol at baseline, which is not
reported in Table 5, and retinol decrease (i.e., retinol at baseline
minus retinol during treatment) were also taken into account.
Only subjects with no evident disease and contralateral or
ipsilateral breast cancer, i.e., the disease conditions with the
highest number of subjects, were considered in the analyses.
When drug levels were correlated with body surface area and
BMI, significant correlations were found only in subjects ⱕ45
years with no evident disease in which 4-HPR levels were
inversely associated with body surface area (r ⫽ ⫺0.38: P ⫽
0.001) and in subjects ⱕ45 years who developed contralateral
or ipsilateral breast cancer in which 4-MPR levels were directly
associated with BMI (r ⫽ 0.61; P ⫽ 0.03; data not shown). A
highly significant correlation was found between 4-MPR and
4-HPR levels (r ⱖ 0.59; P ⱕ 0.03) and between the retinol
decrease and retinol levels at baseline (r ⱖ 0.7; P ⱕ 0.001) in
subjects with no evident disease and in those with contralateral
and ipsilateral breast cancer of all age ranges (data not shown).
4-HPR levels were inversely correlated with retinol levels during treatment (r ⱖ 0.27; P ⱕ 0.02) in subjects with no evident
disease of all age ranges, whereas such a correlation was not
found in subjects who developed contralateral or ipsilateral
breast cancer (data not shown). As the number of subjects with
no evident disease was larger than that of subjects with contralateral or ipsilateral breast cancer, for a more adequate comparison, the same correlation was also assessed on a number of
subjects with no evident disease (randomly selected) equal, for
each age range, to that of subjects with contralateral and ipsilateral breast cancer. The correlation between 4-HPR levels and
retinol levels during treatment was still significant (r ⱖ 0.48;
P ⱕ 0.05; data not shown).
Discussion
The primary objectives of the study were to assess, in subjects
participating in a 4-HPR prevention trial, the influence of age
and menopausal status on drug and retinol plasma levels and
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Cancer Epidemiology, Biomarkers & Prevention
Table 5
4-HPR, 4-MPR, and retinol levels (means ⫾ SD in ng/ml) after 1-year 4-HPR treatment and body surface area and BMI (means ⫾ SD) at baseline
Subjects with
Age (years)
No evident disease
(n)
ⱕ45
46–55
ⱖ56
4-HPR
4-MPR
Retinol
m2b
BMI
4-HPR
4-MPR
Retinol
m2
BMI
4-HPR
4-MPR
Retinol
m2
BMI
(70)
(107)
(57)
394 ⫾ 123.5
387 ⫾ 186.0
152 ⫾ 76.1
1.59 ⫾ 0.12
22.45 ⫾ 3.41
443 ⫾ 156.9c
452 ⫾ 234.9c
177 ⫾ 86.6
1.66 ⫾ 0.13d
24.17 ⫾ 3.77d
427 ⫾ 187.5c
421 ⫾ 231.6c
162 ⫾ 85.0
1.68 ⫾ 0.14d
24.98 ⫾ 4.27d
Contralateral or
ipsilateral breast cancer
(n)
(13)
(19)
(14)
473 ⫾ 172.1a
503 ⫾ 249.9a
109 ⫾ 58.0a
1.60 ⫾ 0.10
22.70 ⫾ 2.59
411 ⫾ 108.2
409 ⫾ 109.5
201 ⫾ 121.7
1.62 ⫾ 0.08
23.61 ⫾ 2.57
419 ⫾ 106.9
406 ⫾ 167.7
168 ⫾ 78.5
1.69 ⫾ 0.15
25.94 ⫾ 4.21
Metastases
(n)
(5)
(19)
(8)
Second primary cancer
(n)
474 ⫾ 177.7
606 ⫾ 418.4
189 ⫾ 97.2
1.66 ⫾ 0.26
24.86 ⫾ 5.94
536 ⫾ 317.1
635 ⫾ 490.9
137 ⫾ 60.1
1.67 ⫾ 0.12
25.78 ⫾ 4.29
512 ⫾ 225.1
525 ⫾ 260.7
160 ⫾ 64.2
1.76 ⫾ 0.05a
28.30 ⫾ 3.66a
(6)
(6)
Toxicity
(n)
377 ⫾ 102.9
322 ⫾ 119.1
190 ⫾ 39.5
1.84 ⫾ 0.21
27.09 ⫾ 5.40
468 ⫾ 234.4
386 ⫾ 85.8
146 ⫾ 101.8
1.57 ⫾ 0.15
23.15 ⫾ 1.42
(3)
366 ⫾ 37.6
408 ⫾ 137.7
106 ⫾ 128.8
1.41 ⫾ 0.03
18.33 ⫾ 0.39
(2)
667 ⫾ 74.2
665 ⫾ 311.4
88 ⫾ 16.9
1.54 ⫾ 0.01
23.40 ⫾ 2.88
P ⱕ 0.05 versus same age patients with no evident disease by Dunnett’s t test.
m2, body surface area.
c
P ⱕ 0.05.
d
P ⱕ 0.005 versus patients ⱕ 45 years, with no evident disease.
a
b
prospectively evaluate their association with a second breast
malignancy.
At randomization, the 4-HPR and control arms had comparable retinol levels. As reported previously by other authors
(15, 16), we found a positive relation between age and retinol
levels. We also found that retinol levels are affected by menopausal status. Among women of the same age range, those in
postmenopause had higher retinol levels than those in premenopause. In addition, among women in the control arm, only
those aged 46 –55 years who were in premenopause at baseline
showed, during the 5-year trial, a significant increase in retinol
levels, which was presumably caused by the change in menopausal status. The reasons for the observed increase in women
with age and after menopause are not known. Several reports
indicate that retinol levels are variously associated with serum
cholesterol and triglycerides (17, 18). The increase in retinol
levels found in older women might be linked to the increase in
circulating lipoproteins that occurs with aging. The results of
the few studies that have explored the influence of sex hormones on retinol plasma levels do not clarify the issue. In fact,
oral contraceptive therapy in young women has been found to
induce a significant increase in retinol levels (19 –21). Although
the reasons for the observed difference are not known, the
findings indicate that age and menopausal status should always
be taken into account when interpreting retinol levels in
women.
Evidence from our and previous studies (6, 8) suggests
that retinol levels are not associated with a risk of breast cancer.
Our results on the role of retinol levels as predictive of breast
tumor recurrence indicate that, in women operated on for stage
I breast cancer, retinol levels have no prognostic value for
contralateral or ipsilateral breast cancer. Less conclusive are the
results on the role of retinol levels in predicting breast cancer
metastasis. Control subjects ⬎56 years who subsequently developed metastasis had lower retinol levels than disease-free
subjects. However, such a difference was not found in subjects
treated with 4-HPR. Additional studies are necessary to clarify
this issue.
Estimates of drug plasma levels demonstrated that a daily
p.o. dose of 200 mg of 4-HPR resulted, at 10 –18 h after
administration, in 4-HPR and 4-MPR plasma levels of ⬃400
ng/ml, corresponding to ⬃1 ␮M. The 4-HPR concentrations
found effective in suppressing the in vitro growth of tumor cells
range from 1 to 10 ␮M (22). Therefore, plasma levels of 1 ␮M
4-HPR correspond to the least effective concentrations in in
vitro models. However, we showed previously that 4-HPR
accumulates in the breast because its concentrations in nipple
discharge were 10 times higher than those in plasma (4). 4-HPR
and 4-MPR levels, which were directly related, remained constant during the 5-year treatment. The results confirm previously reported data found in a small group of women participating in a Phase I trail (4).
With regard to the possible influence of age and menopausal status on 4-HPR pharmacokinetics, the present data
indicate that circulating 4-HPR and 4-MPR levels are not linked
to menopausal status but that they are influenced by patient age.
After identical daily doses during 1 year of treatment, subjects ⱕ 45 years had slightly, but significantly, lower 4-HPR
and 4-MPR plasma levels than the older subjects. This occurred
despite the marked lower body surface area of women ⱕ 45
years than those in the other age ranges. Lower drug absorption,
higher metabolism to undetected metabolites, or higher biliary
excretion might account for the lower 4-HPR and 4-MPR levels
found in the youngest women. After their absorption from the
gastrointestinal tract, retinoids, which are water insoluble compounds, bind to serum albumin and lipoproteins and are transported to the tissues. Although acidic retinoids, like all-trans
retinoic acid, bind preferentially to albumin, neutral retinoids
bind to lipoproteins (23). We do not know how 4-HPR is
transported in plasma, but because 4-HPR is a neutral retinoid,
it should be carried by lipoproteins. Therefore, the increased
plasma levels of 4-HPR in older subjects might be attributable
to the drug binding to concurrent plasma lipoproteins, whose
levels usually increase with increasing age.
Unexpectedly, we found that subjects ⱕ 45 years with
breast cancer recurrence had slightly, but significantly, higher
drug levels than subjects with no evident disease. Unfortunately, because the sample size in the two groups was unbal-
Downloaded from cebp.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research.
39
40
Fenretinide Plasma Levels and Second Breast Malignancy
anced and the difference was small, we cannot draw conclusions on the role of drug plasma levels as predictive of 4-HPR
efficacy. However, the results, in conjunction with the finding
of higher drug plasma levels in older subjects who did not show
any preventive benefit from 4-HPR treatment (1), suggest an
inverse association between plasma drug levels and 4-HPR
preventive effects. An explanation for this intriguing result
might be that higher drug plasma levels reflect lower drug
concentrations in the tissues where they are needed. It may be
that the binding of 4-HPR to lipoproteins in older women and
young nonresponding women might inhibit entry of the drug
into cells. In vitro data on tumor cell lines support such a
hypothesis. In in vitro assays, the addition of retinoids using
serum-free conditions or low serum concentrations resulted in
dose-dependent growth inhibitory effects, which decreased
when serum or serum components were added (24 –26). It has
also been demonstrated that reduced growth inhibitory effects
were associated with decreased retinoid uptake by the cells
(25). The same applies to the in vitro growth inhibitory effects
of 4-HPR.4
Although the difference was not statistically significant,
subjects who subsequently developed metastases also had
higher 4-HPR and 4-MPR levels than subjects with no evident
disease. Moreover, a high percentage of these subjects had
abnormally high (ⱖ900 ng/ml) 4-MPR plasma levels. 4-HPR
and 4-MPR are both lipophilic, and 4-MPR has a longer storage
than 4-HPR in deep tissue compartments, presumably fat, from
which it is slowly released (4). The abnormally high 4-MPR
concentrations found in subjects who subsequently developed
metastasis presumably reflect a high percentage of adipose
tissue in these subjects.
The results of the 4-HPR breast tumor prevention trial
have shown the good tolerability of the retinoid at 200 mg/day
over a 5-year treatment (1, 12). The present results indicate that
the few subjects who experienced 4-HPR toxicity and whose
mean drug plasma levels are herein reported had a smaller body
surface area than subjects who showed no toxicity. The data
suggest that 4-HPR should be administered on the basis of body
surface area. Moreover, 4-HPR toxicity was associated with
low retinol levels before 4-HPR administration but only in
subjects aged ⱕ45 years. This age group had the lowest pretreatment retinol levels, and the additional reduction caused by
4-HPR in these subjects may have resulted in retinol levels
below threshold limits.
Regarding the effect of 4-HPR on retinol plasma levels,
we had shown previously that 4-HPR reversibly reduces the
plasma concentrations of retinol and of its transport protein,
RBP (4, 5). We also showed that the high binding affinity of
4-HPR for RBP and the lack of binding of the complex 4-HPRRBP to transthyretin (27) might account for the reduction of
retinol secretion from retinol-secreting organs. Retinol levels
after a 5-year treatment were always slightly, but significantly,
lower than those found at 1 year. The difference, although it
does not seem to be biologically relevant, might indicate 4-HPR
accumulation in retinol-secreting organs like the liver. A new
finding was that the reduction in retinol levels was strongly and
directly correlated with pretreatment retinol levels. Therefore,
pretreatment retinol levels may predict retinol level reduction
by 4-HPR; in fact, the higher the initial values, the higher the
absolute decrease, with the percentage decrease remaining the
same. Another interesting observation was that the inverse
4
Formelli et al., manuscript in preparation.
correlation between 4-HPR and retinol levels during treatment
was significant in subjects with no evident disease, whereas it
was not significant in those in whom breast cancer recurred.
This result seems to further support the evidence of differences
in 4-HPR pharmacokinetics between subjects with no evident
disease and those who did not benefit from 4-HPR treatment.
Interestingly, differences in retinoid metabolism have been
found between human tumors and the respective normal tissues
(28).
In conclusion, in a prospective study in women participating in a breast tumor prevention trial with 4-HPR, we found that
subjects ⱕ 45 years had lower drug plasma levels than older
subjects, and, among young subjects, those with breast cancer
recurrence had higher drug levels. A tentative explanation for
this unexpected finding could be related to drug interaction with
serum proteins, drug sequestration in plasma, and consequent
interference with the tumor preventive potential. The hypothesis of serum components as determinant of 4-HPR plasma
levels and bioavailability should be investigated in future studies with the retinoid.
Acknowledgments
We thank Dr. Carmen Pollini for her help in data analysis and critical discussion
of the results and Laura Zanesi for secretarial assistance.
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41
Fenretinide Breast Cancer Prevention Trial: Drug and
Retinol Plasma Levels in Relation to Age and Disease
Outcome
Franca Formelli, Tiziana Camerini, Elena Cavadini, et al.
Cancer Epidemiol Biomarkers Prev 2003;12:34-41.
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