Biomedicine & Pharmacotherapy 59 (2005) 253–263
http://france.elsevier.com/direct/BIOPHA/
Original article
Beta-interferon and interleukin-2 prolong more than three times
the survival of 26 consecutive endocrine dependent breast cancer patients
with distant metastases: an exploratory trial
Andrea Nicolini a,*, Angelo Carpi b
a
Department of Internal Medicine, University of Pisa, Via Roma 67, 56126 Pisa, Italy
b
Department of Reproduction and Aging, University of Pisa, Pisa, Italy
Received 16 April 2004; accepted 12 May 2004
Available online 04 May 2005
Abstract
Distant metastases from breast cancer are incurable. In endocrine-responsive patients antiestrogens are commonly administered as first and
second line therapy. Regrettably, tumor growth becomes resistant to this relatively innocuous therapy. Beta-interferon was unsuccessfully
added to tamoxifen to induce estrogen receptor enhancement. In mice, interleukin-2 added to tamoxifen increased their mutual anti-tumor
activities. Nevertheless, no effective clinical application has been developed. We started an exploratory clinical trial based on the association
of these immunostimulating cytokines with antiestrogens for first line salvage therapy of hormone dependent breast cancer with distant
metastases. Twenty-six consecutive breast cancer patients with distant metastases, 23 of which had metastases at multiple sites, were studied
for responsiveness to treatment with first line salvage antiestrogen therapy, combined with beta-interferon and interleukin-2 immuno-therapy.
Clinical response and survival were compared with that of 30 consecutive historical control patients treated with antiestrogen therapy alone.
Controls showed, as expected, a median duration of response, a median survival time after treatment, and after diagnosis of distant metastases,
of 16, 31 and 34 months, respectively. After a mean follow-up of 62 ± 36 months (range 17–155), the interval times in the non-control patients
were 61 (P < 0.001), 101 (P < 0.000001) and 106 (P < 0.000001) months. Two long-term survivors appeared to be cured after 155 and
94 months from the time of diagnosis with multiple bone metastases. Nineteen of the patients treated with beta-interferon and interleukin2 have survived. Hormone immuno-therapy was given in an outpatient setting and was very well tolerated. These data suggest that immunotherapy plays an important role in endocrine-dependent metastatic breast cancer.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Breast cancer; Distant metastases; Immuno-therapy
1. Introduction
Distant metastases from breast cancer are incurable [4]. In
endocrine-responsive patients antiestrogens are commonly
administered as first and second line therapy [8,23]. Regrettably, tumor growth becomes resistant to this therapy and usually heralds disease progression. Many mechanisms have been
considered to explain this resistance [44]. Following experimental studies [35,36] beta-interferon was unsuccessfully
[6,32] added to tamoxifen to induce estrogen receptor
enhancement. In mice, interleukin-2 added to tamoxifen
increased their mutual anti-tumor activities [17]. Neverthe* Corresponding author. Tel.: +39 050 99 2141; fax: +39 050 55 3414.
E-mail address: [email protected] (A. Nicolini).
0753-3322/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.biopha.2004.05.019
less, no effective clinical application has been developed. In
man these cytokines are well tolerated in low dose regimens
[2,32]. In 1992 we decided to start an exploratory clinical
trial based on the association of these immunostimulating
cytokines [13,34] with antiestrogens for first line salvage
therapy of hormone dependent breast cancer with distant
metastases.
2. Materials and methods
2.1. Patient recruitment
Our practice in metastatic patients was to sistematically
test first line antiestrogen salvage therapy. All patients respon-
254
A. Nicolini, A. Carpi / Biomedicine & Pharmacotherapy 59 (2005) 253–263
Table 1
Details of the patients ’ recruitment
Study groups
Recruitment interval
Pts with distant
metastases who came
to observation
Pts who withdrew from
the follow-up
Pts who received antiestrogen first line salvage treatment
n
responsive or with SD
n
%
%
n
%
16
31.5
31.5
20
Total
12
23
23
15
73
2
6
5
7
20
17
26
22
47
27
10
17
18
8
53
5
7
8
3
23
50
41
44
37.5
43
5
10
10
5
30
50
59
56
62.5
57
32
26
22
20.5
Total
25
20
17
16
78
7
5
2
3
17
28
25
12
19
22
18
15
15
13
61
11
10
7
7
35
61
67
47
54
57
7
5
8
6
26
39
33
53
46
43
Controls
Oct 81–Sep 84
“ 84–“ 87
“ 87–“ 90
“ 90–Mar 92
Exploratory study
Apr 92–Mar 95
“ 95–“ 98
“ 98–“ 01
“ 01–Apr 03
sive, or with stable disease (SD) on tamoxifen were treated at
the Department of Internal Medicine, Pisa University from
1981 to 1992 with first line tamoxifen only (historical control) and from 1992 to 2003 with tamoxifen or toremifene
plus immuno-therapy (ongoing exploratory study group). The
details of the recruitment are reported in Table 1.
Historical control. Among 73 non-selected breast cancer
patients observed from October 1981 to March 1992, 20
(27%) withdrew from the follow-up. Twenty-three (43%) of
the 53 remaining showed no clinical benefit from first line
salvage therapy with tamoxifen and the historical controls
were the 30 others (57%) with SD, partial or complete
response (CR) during therapy with tamoxifen.
Ongoing exploratory study group. Among 78 non-selected
breast cancer patients observed from April 1992 to April 2003,
17 (22%) withdrew from the follow-up. Thirty-five (57%) of
the 61 remaining showed no clinical benefit from first line
antiestrogens and the 26 others (43%) with SD or responsive
to first line antiestrogens, were consecutively recruited for
the addition of immuno-therapy.
In particular, the first 12 patients recruited from April
1992 to March 1998 and another recruited in November
1999 were under tamoxifen when they were given immunotherapy. The successive 13 recruited from April 1998 to April
2003, received toremifene associated with immuno-therapy.
Tamoxifen was replaced with toremifene [14] for higher safety
[12] in 1998 when it was provided by the National Health
Service.
In the study group the time for the assessment of a disease
stable, or responsive to hormone therapy, before immunotherapy, was defined as induction time, and had to be a minimum of 2 months.
All 26 patients gave witnessed written informed consent
and the study was approved by the Council of the Department of Internal Medicine of Pisa University.
n
in progression
%
n
2.2. Inclusion and exclusion criteria
For both historical controls and the study group the inclusion criteria were the following: operated breast cancer
patients of any age with distant metastases (M1) stable or
responsive to first line antiestrogen salvage therapy. At entry,
an ECOG performance status of 0 to 3, adequate hematopoietic, cardiac, renal and liver function and the ability to undergo
regular clinical-radiological monitoring were required.
For both control and study group the exclusion criteria was
a previous or concomitant malignancy without high probability of definite cure. For the study group need of corticosteroids was an exclusion criteria.
2.3. Treatment program and study design
2.3.1. First line salvage therapy
Historical controls. Tamoxifen was continuously administered 20 mg daily dose b.m. to the progression of metastatic
disease.
Study group (Fig. 1) During the induction time and successively, tamoxifen (1992–1999, 20 mg/day) or toremifene
(1998–2002, 60 mg/day) was given continuously. After the
induction time beta-interferon 3,000,000 IU i.m./day was
added 3 days/week for 1 month (weeks 1–4). In these 4 weeks
the daily dose of tamoxifen and toremifene was increased to
30 and 90 mg, respectively, because in vitro [16] and in vivo
[28] studies suggested an up regulation of estrogen receptors
by tamoxifen in human breast cancer. Successively, recombinant interleukin-2 3,000,000 IU s.c./day (3 days a week)
for 4 weeks was added (weeks 5–8). The patient continued
antiestrogen therapy only during weeks 9–12. The cycle lasted
12 weeks (3 months), and was then repeated, the 13th week
being the first week of the successive cycle. At each cycle,
5 days before the recombinant interleukin-2 and during its
administration, melatonin 40 mg/day b.m., at 8.30–9 p.m. was
given because it enhanced clinical efficacy of recombinant
A. Nicolini, A. Carpi / Biomedicine & Pharmacotherapy 59 (2005) 253–263
255
Fig. 1. Schedule of first line hormone-immunotherapy.
Note. In patients who had second line hormone-immunotherapy, tamoxifen or toremifene was replaced by letrozole at the constant 2.5 mg daily dose.
interleukin-2 [21], stimulated estrogen receptor expression
on breast cancer cells [7] and reduced cachexia in advanced
cancer patients [22].
2.3.2. Second line salvage therapy
Historical controls. In all 30 controls when resistance to
tamoxifen developed, medroxyprogesterone acetate was
administered 2 g daily dose b.m. for the first 20–30 days, and
successively 1 g daily.
Study group Tamoxifen or toremifene were replaced with
letrozole, 2.5 mg/day. Patients receiving letrozole after progression of metastatic disease again were assessed for 2 or
more months (induction time). In the patients responsive or
with SD but two, the same schedule of immuno-therapy was
adopted; the daily dosage of letrozole was not increased concomitantly with beta-interferon, because no study documented an up-regulation of estrogen receptors by letrozole.
In 1 of the 2 remaining letrozole was not yet available when
resistance to tamoxifen developed and the other, after first
line chemo and hormone-immuno salvage therapy, refused
any treatment for injection.
Immuno-therapy during first and second line hormonetherapy was self-administered in an outpatient setting.
2.3.3. Other treatments
In the control and study groups, one regimen of chemotherapy was given to a few patients before first line hormone
therapy (Table 2) and bisphosphonates were administered to
all patients with lytic or mixed (lytic and blastic) bone metastates.
2.3.4. Study design
The relatively low recruitment rate of our Center led us to
carry out an initial exploratory clinical trial and to use historical controls for comparison. In our Center no important
change in the recruitment of the metastatic patients had
occurred from 1981 to 2002. Furthermore from 1981 up to
the present median survival of these patients submitted to the
conventional treatments did not change significantly [9,29].
The study intended to evaluate possible clinical benefit (duration of stable or responsive disease and overall survival), drug
toxicity and assessable changes of some immunological
parameters. The last post-operative follow-up observation was
June 30, 2003.
2.3.5. Treatment assessment and tolerability
of hormone-immunotherapy
Historical control and study group. Evaluation of CR,
partial response (PR), SD (i.e. no change) or progressive disease (PD) followed modified World Health Organization criteria [23]. Clinical benefit included CR, PR and SD. Performance status was assessed by the ECOG scale. Metastatic
disease progression was suspected and ascertained following
previously reported criteria [27]. In the study group, an initial treatment assessment was carried out at the end of the
induction time. Successively, the best clinical result lasting at
least 24 weeks defined the clinical benefit (CR, PR or SD). In
256
A. Nicolini, A. Carpi / Biomedicine & Pharmacotherapy 59 (2005) 253–263
Table 2
Metastatic breast cancer: main characteristics of consecutive patients who received only antiestrogen (tamoxifen, 1981–1992) or antiestrogen plus immunotherapy (tamoxifen, 1992–1999 or toremifene, 1998–2003) as first line treatment
Characteristic
Group on antiestrogen
Tamoxifen
(1981–1992)
n = 30
pts (%)
Ag(years)
m ± S.D.
62.3 ± 12.5
Range
37–84
ECOG performance status
0; 1
30
Menopause, n(%)
Pre
4 (13%)
Post
26 (87%)
Receptor status, n(%)
ER+ PR+
6 (20%)
ER+ PR–
6 (20%)
ER– PR+
3 (10%)
ER– PR–
2 (7%)
Unknown
13 (43%)
(ER+, all cases)
12 (40%)
(PR+, all cases)
9 (30%)
Disease-free interval, n(%)
Months
< 24
6 (20%)
≥ 24
24 (80%)
Distant metastases, n(%)
Site
Bone
15 (50%)
Visceral
5 (17%)
Soft tissue
4 (13%)
Bone + soft tissue
1 (3%)
Bone + visceral
3 (10%)
Bone + visceral + soft tissue
0
Visceral + soft tissue
2 (7%)
(Soft tissue, all cases)
7 (23%)
4 (13%)
(Liver§, all cases)
Lesions, number, n(%)
1
10 (33%)
2
5 (17%)
≥3
15 (50%)
Adjuvant therapy, n(%)
Chemotherapy
10 (33%)
Tamoxifen(months)
< 12
3 (10%)
≥ 12
12 (40%)
Other salvage therapy before antiestrogens, n(%)
Radiotherapy and/or chemotherapy
3 (10%)
Tamoxifen
(1992–1999)
n = 13
pts (%)
Groups on antiestrogen plus immuno-therapy
Toremifene
Either
(1998–2002)
(1992–2002)
n = 13
n = 26
pts (%)
pts (%)
58.6 ± 8.8*
47–70
65.5 ± 13.9*
33–82
62 ± 12*
33–82
13
13
26
3 (23%)†
10 (77%)
1 (8%)†
12 (92%)
4 (15%)†
22 (85%)
6 (46%)
0
0
2 (15%)
5 (39%)
6 (46%)†
6 (46%)†
3 (23%)
5 (39%)
0
2 (15%)
3 (23%)
8 (62%)†
3 (23%)†
9 (35%)
5 (19%)
0
4 (15%)
8 (31%)
14 (54%)†
9 (35%)†
1 (8%)†
12 (92%)
2 (15%)†
11 (85%)
3 (12%)†
23 (88%)
7 (54%)
1 (8%)
0
2 (15%)
2 (15%)
0
1 (8%)
3 (23%)†
2 (15%)†
3 (23%)
3 (23%)
1 (8%)
0
2 (15%)
1 (8%)
3 (23%)
5 (38%)†
2 (15%)†
10 (39%)
4 (15%)
1 (4%)
2 (8%)
4 (15%)
1 (4%)
4 (15%)
8 (31%)†
4 (15%)†
1 (8%)
3 (23%)
9 (69%)†
2 (15%)
2 (15%)
9 (69%)†
3 (12%)
5 (19%)
18 (69%)†
4 (31%)†
7 (54%)†
11 (42%)†
1 (8%)
5 (38%)†
0
7 (54%)†
1 (4%)
12 (46%)†
4 (31%)†
5 (38%)‡
9 (35%)‡
Compared with controls (only with antiestrogen) *P n.s., unpaired t-test; †P n.s., ‡P < 0.05, Fisher ’s exact probability test; §among the patients with visceral
metastases. ER, PR refer to primary tumour; they were measured by immunohistochemical assay (cut-off > 10% immunopositive malignant cells) or by dextran
coated charcoal method (cut-off 10 fmol/mg of cytosol protein).
the study group adverse event was defined as any detrimental
change in the patient ’s condition after the beginning of the
hormone immuno-therapy unless considered to be related to
disease progression. Any adverse event was recorded irrespective of whether it was considered related to the trial
therapy. Severity of adverse events was graded by National
Cancer Institute ’s common toxicity criteria (version 2.0).
2.4. Statistical analysis
At basal the comparison between the 30 controls and the
first 13, the 13 successively recruited and all the 26 patients
of the ongoing study was carried out by the unpaired t-test
for age and by Fisher ’s exact test for other principal characteristics.
A. Nicolini, A. Carpi / Biomedicine & Pharmacotherapy 59 (2005) 253–263
In the 30 controls, the first 13, the 13 successively recruited,
and all the 26 studied patients, clinical benefit and overall
survival curves from first line antiestrogen or from the relapse
were estimated by the Kaplan–Meier method [24]. The same
method was used to estimate the duration of response (CR
and PR) in the 30 controls and in the 26 studied patients.
Differences between curves were tested by the log-rank
test. Relapse time was that of the first pathological finding
ascertained or suspected.
A Cox uni and multivariate analysis was performed [38]
with the following variables: type of the treatment, age (continuous values), menopause (pre or post), estrogen and progesterone receptors (positive or negative and unknown), diseasefree interval (< 24 or ≥ 24 months), soft tissue and liver
metastatic involvement (with or without), the number of metastatic lesions (≥ 3 or < 3), adjuvant chemotherapy (with or
without), adjuvant tamoxifen (≥ 12 or < 12 months) radiotherapy and/or chemotherapy before antiestrogen salvage
therapy (with or without), presence of two or more negative
predictive factors (≥ 3 lesions, soft tissue or liver involvement, disease-free interval < 24 months and adjuvant chemotherapy).
3. Results
3.1. Historical and study groups: clinical details,
response, clinical benefit and overall survival
In the two subgroups of 13 patients of the study group,
and in all these 26 patients the main characteristics at relapse
were not significantly different from the 30 controls (Table 2).
Tables 3 and 4 show the main clinical characteristics and
therapeutic events of these 26 patients treated with antiestrogen plus immuno-therapy.
In the 9 (30%) responsive controls the median response
was 16 months and in the 12 (46%) responders of the study
group it is currently 61 months (P < 0.001). However, in the
study group this time is not definite because seven (58%) of
the 12 responders are still in response and four have a relatively short follow-up (Table 4). Two (15%) of the first
13 patients treated long-term with hormone immuno-therapy
are still in CR after 149 and 94 months (Table 4).
As to the clinical benefit, highly significant difference
between the 30 controls and all 26 studied patients occurred
(P < 0.000001) (Fig. 2a). After 12 months the patients on clinical benefit were 57% in the controls while they were 100% in
the study group. At 31 months no control showed any benefit
while 62% of the studied patients still maintained a benefit.
Successively, in this group the clinical benefit progressively
decreased reaching 13.6% at 70 months (Fig. 2a). The loss of
the clinical benefit up to 30 months in the controls was much
faster than in the two subgroups of the study group (first subgroup, n = 13, 1992–1999, vs. controls, P < 0.000001; second subgroup, n = 13, 1998–2002, vs. controls, P < 0.0001).
In these two subgroups the clinical benefit was very similar
257
and at 30 months no significant difference occurred (84.6%
vs. 68.8%) (Fig. 2b).
Overall survival from first line antiestrogen showed a
highly significant difference in the 30 controls compared with
all 26 studied patients (P < 0.000001) (Fig. 2c). Survival from
antiestrogen after 30 months was 50% in the 30 controls while
it was 100% in the 26 patients of the study group. At
78 months no historical control survived and 73% of the study
group was alive. Successively in these latter patients the proportion progressively decreased reaching 22.7% at
120 months (Fig. 2c). The death rate up to 60 months was
much faster in the controls than in the two subgroups of the
study group (first subgroup vs. control, P < 0.0001; second
subgroup vs. control, P < 0.05). The two subgroups showed
a similar survival (75.5% and 75%, at 60 months, P n.s.)
(Fig. 2d).
Also overall survival from distant metastases showed an
highly significant difference in the 30 controls compared with
all 26 studied patients (P < 0.000001) (Fig. 2e). Survival at
30 months from the first distant metastasis was about 57% in
the controls while it was 100% in the study group. At
90 months no control survived and 74% of the study group
were alive. Successively in these latter patients survival progressively decreased reaching 21.2% at 120 months (Fig. 2e).
The death rate up to 60 months was much faster in the controls than in the two subgroups of the study (first subgroup
vs. control, P < 0.0001; second subgroup vs. control,
P < 0.01). The two subgroups showed a similar survival
(92.3% and 83.3% at 60 months, P n.s.) (Fig. 2f).
The Cox univariate analysis showed that the type of treatment significantly affected clinical benefit and overall survivals. The Cox multivariate analysis showed that adjusting for
covariates, the type of treatment was highly significant with
regard to the clinical benefit at 96 (Fig. 2a, P < 0.000001) and
at 30 months (Fig. 2b, P < 0.00001), the overall survival from
first line antiestrogen at 120 (Fig. 2c, P < 0.00001) and at
60 months (Fig. 2d, P < 0.001) and the overall survival from
distant metastases at 120 (Fig. 2e, P < 0.00001) and at
60 months (Fig. 2f, P < 0.001).
3.2. Tolerability of hormone immuno-therapy (Table 5)
No death occurred during this treatment and no event led
to interruption of therapy. In two patients (n. 4 and n. 10)
fever > 39 °C decreased considerably or disappeared by dividing interleukin-2 dose in two halves given morning and
evening. In patients n. 13 and n. 15, aged 69 and 80 years,
increasing creatininemia (to 3.15 and to 2.66 mg/dl respectively) was halted by reducing up to 75% the number of betainterferon and interleukin-2 administrations per cycle, according to creatinine clearance value.
4. Discussion
Our patients on clinical benefit during first line antiestrogen therapy and treated with the addition of immuno-therapy
Age
(years)
Menopause
Receptor status (fmol/mg)
Progesteron
*87
unknown
Disease-free
interval
(months)
0
138
Distant
metastases
(date)
90-07-27
91-12-19
1
2
47
65
pre
post
Estrogen
*21
unknown
3
70
post
unknown
unknown
138
93-03-18
4
58
post
*22
*96
90
93-08-26
5
6
7
8
52
69
68
66
pre
post
post
post
†negative
†70%; +++
unknown
unknown
†negative
†60%; ++
unknown
unknown
61
55
142
80
94-04-11
95-01-02
92-09-08
94-11-15
9
10
11
50
64
57
post
post
post
*26
*64
†20%; +++
*93
*41
†20%; ++
59
104
122
95-08-17
95-12-01
95-02-03
12
13
14
48
62
74
pre
post
post
†negative
unknown
*13.7
†negative
unknown
*negative
47
93
107
96-06-13
96-11-12
98-08-15
15
77
post
†80%; +++
†negative
0
99-05-05
16
56
post
*22.6
*negative
177
00-03-31
17
82
post
unknown
unknown
341
00-09-05
18
19
69
61
post
post
†70%; +++
unknown
†70%; +++
unknown
48
216
00-08-30
00-09-25
20
58
post
†‡85%; +++
†‡15%, +
149
00-10-24
21
22
23
24
79
52
58
80
post
post
post
post
†40%; +
†negative
*neg
unknown
†negative
†negative
*neg
unknown
117
45
85
147
01-06-23
01-04-19
98-09-28
01-11-27
25
71
post
†80%; +++
†negative
63
01-09-15
26
34
pre
†85%; ++
†30%; +
0
02-01-21
Site
(organ)
Lesion
(n)
bone
bone
liver
pleural effusion
bone
lung
subcarinal and right
hylar lymph nodes
bone
bone
bone
bone
mediastinal lymph-nodes
bone
liver
bone
skin
supraclavicular lymph nodes
bone
bone
cutaneuos lymphangytis
mediastinal lymph-nodes
bone
lung
internal mammary nodes
bone
pleural effusion
liver
peritoneal carcinomatosis
pleural effusion
lung
pleural effusion
lung
supraclavicular lymph-nodes
bone
bone
bone
lung
pleural effusion
hylar bilateral lymph-nodes
subcarinal lymph-nodes
lung
pleural effusion
bone
supraclavicular lymph-nodes
lung
liver
2
2
1
1
>3
3
2
2
1
>3
1
1
>3
>3
2
1
1
>3
3
1
1
2
>3
1
>3
1
>3
1
1
>3
1
1
1
1
>3
>3
>3
1
2
1
>3
1
2
1
2
>3
Adjuvant
tamoxifen
(months)
no
>12
Rt and /or Ct before
salvage therapy with
antiestrogen
Ct
–
12
–
4
–
no
>12
no
no
–
–
–
–
no
>12
no
–
Ct
Ct and Rt
no
>12
>12
Ct and Rt
–
–
no
Ct
>12
Ct
no
–
>12
>12
–
–
no
–
>12
no
>12
no
–
Ct
Ct
–
>12
–
no
Ct
*Dextran coated charcoal method: †immunohistochemical method (immunostaining: + weak, ++ moderate, +++ strong); ‡ = receptor determination in metastatic lung nodule.
A. Nicolini, A. Carpi / Biomedicine & Pharmacotherapy 59 (2005) 253–263
Patient
(n)
258
Table 3
Main characteristics of 26 consecutive hormone-responsive metastatic breast cancer patients treated with tamoxifen (from n. 1 to n. 13, years 1992–1999) or toremifene (from n. 14 to n. 26, years 1998–2003) plus
beta-interferon-interleukin-2 as first line salvage therapy
Table 4
Clinical benefit on first line salvage therapy with tamoxifen or toremifene, clinical course on second line salvage therapy with letrozole and overall survival in 26 consecutive hormone- responsive metastatic breast
cancer patients treated with the addition of beta-interferon and interleukin-2 immuno-therapy
Patient (n)
First line salvage therapy with tamoxifen or toremifene plus
immuno-therapy
Date
Induction time
Clinical benefit
(months)
Type
Months
+
91-02-05
93-01-21
93-04-29
14
7
6
CR
SD
PR
149
47
33
4
5
93-09-28
94-09-27
4
3
PR
SD
61
30
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
95-01-02
92-09-10
95-07-15
95-08-29
96-05-17
97-10-25
97-11-08
99-07-07
98-11-25
99-10-02
00-07-25
00-09-07
00-09-22
00-12-20
00-11-29
3
32
2
5
6
2
10
5
6
4
3
3
4
3
6
21
22
23
24
25
26
01-07-27
01-12-10
01-11-16
01-11-28
02-03-21
02-07-01
4
3
7
3
5
3
SD
SD
PR
CR
SD
SD
SD
PR
PR
SD
PR
SD
SD
PR
SD
(stage IV
NED)
PR
SD
SD
PR
PR
SD
Date
Induction time
(months)
Clinical course
Overall survival
Type
Months
–
2
2
–
SD
P
–
21
–
From
antiestrogen
(months)
149++
101
60
From relapse
(months)
PS
155++
114
60
0
dead
dead
2
3
SD
P
20
–
105
58
106
63
dead
dead
31
70
17
94+
41
45
54
48+
16
33
31
31
28
30+
31+
–
98-12-16
97-12-27
(after MPA)
99-01-18
98-12-15
(after MPA)
98-11-25
98-07-24
not given
–
99-10-15
–
not given
–
00-10-25
02-07-09
03-04-13
03-04-03
03-04-12
–
–
2
2
–
–
2
–
–
–
3
3
–
–
–
–
–
SD
SD
–
–
PR
–
–
–
PR
SD
–
P
–
–
–
22
23
–
–
27
–
–
–
29+
9+
–
–
–
–
–
102++
94
31
94++
85++
68++
68++
48++
55++
45++
35++
33
33++
30++
31++
102++
94
33
94++
90++
101++
85++
80++
58++
50++
39++
33
34++
33++
32++
3
dead
dead
0
2
2
3
0
0
0
0
dead
0
0
0
23+
13
19+
19+
15+
12+
–
03-04-15
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
23++
19++
19++
19++
15++
12++
24++
26++
57++
19++
21++
17++
0
0
0
0
0
0
A. Nicolini, A. Carpi / Biomedicine & Pharmacotherapy 59 (2005) 253–263
1
2
3
Second line salvage therapy with letrozole plus immuno-therapy
Abbreviations: SD = stable disease; PR = partial response; CR = complete response; P = progression; PS = ECOG performance status; MPA = medroxyprogesterone acetate; NED = no evidence of disease; +in
clinical benefit; ++alive.
259
260
A. Nicolini, A. Carpi / Biomedicine & Pharmacotherapy 59 (2005) 253–263
Fig. 2. Clinical benefit from first line antiestrogen in the 30 controls (______) vs. all 26 studied patients (-.-.-.-) (a) and vs. the two subgroups (on tamoxifen-----,
P < 0.000001; on toremifene - - - - - - - - , P < 0.0001) (b). Overall survival from first line antiestrogen in the 30 controls (_______) vs. all 26 studied patients
(-.-.-.-) (c) and vs. the two subgroups (on tamoxifen ----, P < 0.0001; on toremifene -------- , P < 0.05) (d). Overall survival from distant metastases in the
30 controls (_______) vs. all 26 studied patients (- . - . - . -) (e) and vs. the two subgroups (on tamoxifen - - - - - , P < 0.0001; on toremifene - - - - - - - - , P < 0.01)
(f). Clinical benefit and overall survivals between the two subgroups was never significantly different. P values concerning comparisons shown in b, d and f were
adjusted according to Bonferroni method.
showed median response, median duration of clinical benefit
and median survival at least three times longer than historical
controls and literature series. In fact in most recent relevant
studies, the median duration of response (CR + PR) from
tamoxifen given as first line salvage therapy ranged from
15.1 to 23 months [4,5,10,14,15,26,41]. These values were
similar to the value of our controls (16 months). Accordingly,
median overall survival from first line tamoxifen ranged from
15 to 38.5 months in literature studies [5,10,14,15,25,31,41]
and was 31 months in our controls. Benefit and response following first line antiestrogen treatment mainly depend on the
positive estrogen receptor status [36,40]. In all these series
(literature studies, our control and ongoing study group) the
proportion of hormone dependent patients was similar (ranging from 40% to 70%). Therefore the duration of response
and clinical benefit from first line antiestrogen therapy of these
series were probably comparable. In the last two decades
median survival of metastatic patients did not significantly
change in spite of new available options including second
line hormone therapy [29]. Accordingly median survival of
those who benefit from first line salvage endocrine treatment
is not significantly different from that of the whole population of metastatic patients [9,37]. Indeed some studies
[4,5,10,26,31,41] included 10% to more than 50% patients
A. Nicolini, A. Carpi / Biomedicine & Pharmacotherapy 59 (2005) 253–263
261
Table 5
Predefined and other adverse events during hormone immuno-therapy with cyclic beta-interferon-interleukin-2 in addition to antiestrogens (tamoxifen, toremifene or letrozole)
Adverse event
Predefined
Palpitations
Cardiac ischemia
Dyspnea
Coughing
Anorexia
Dyspepsia
Nausea
Vomiting
Constipation
Dizziness
Headache
Insomnia
Hot flashes
Vaginal bleeding
Laboratory exams
Hypercreatininemia
Elevated AST
Elevated ALT
Elevated cGT
Elevated alkaline phosphatase
Anemia
Thrombocytopenia
Leukopenia
Injection site reaction
Flu-like syndrome
Fever
Asthenia
Myalgia
Arthralgia
Other
Asthmatic syndrome
Hypoalbuminemia
Flatulence
First line hormone immuno-therapy
Patients on tamoxifen (n = 13) (follow-up
Patients on toremifene (n = 13)
52 ± 29 months, m ± S.D.) (range
(follow-up 20 ± 8 months, m ± S.D.)
18–132)
(range 8–30)
Grade 1–2 a
Grade 0–1 a
Grade 1–2 a
Grade 0–1 a
Second line hormone immuno-therapy
Patients on letrozole (n = 7) (follow-up
21.5 ± 9 months, m ± S.D.) (range 6–34)
n
1
0
0
2
2
1
3
3
0
1
0
0
1
3
(%)
(8)
(0)
(0)
(15)
(15)
(8)
(23)
(23)
(0)
(8)
(0)
(0)
(8)
(23)
n
0
1
0
0
1
1
1
0
0
0
1
0
0
0
(%)
(0)
(8)
(0)
(0)
(8)
(8)
(8)
(0)
(0)
(0)
(8)
(0)
(0)
(0)
n
0
0
0
1
4
1
4
3
1
0
1
1
0
0
(%)
(0)
(0)
(0)
(8)
(33)
(8)
(33)
(23)
(8)
(0)
(8)
(8)
(0)
(0)
n
0
0
0
0
1
0
0
0
0
0
0
0
0
0
(%)
(0)
(0)
(0)
(0)
(8)
(0)
(0)
(0)
(0)
(0)
(0)
(0)
(0)
(0)
n
0
0
0
1
1
0
0
0
1
1
0
2
0
0
(%)
(0)
(0)
(0)
(14)
(14)
(0)
(0)
(0)
(14)
(14)
(0)
(28)
(0)
(0)
n
0
0
1
1
0
0
0
0
1
0
0
0
0
0
(%)
(0)
(0)
(14)
(14)
(0)
(0)
(0)
(0)
(14)
(0)
(0)
(0)
(0)
(0)
0
3
3
6
0
(0)
(23)
(23)
(46)
(0)
3
0
1
4
0
(23)
(0)
(8)
(31)
(0)
1
2
1
8
0
(8)
(17)
(8)
(61)
(0)
1
0
0
1
0
(8)
(0)
(0)
(8)
(0)
2
0
0
1
2
(28)
(0)
(0)
(14)
(28)
1
0
0
1
0
(14)
(0)
(0)
(14)
(0)
8
2
2
6
(62)
(15)
(15)
(46)
0
0
0
7
(0)
(0)
(0)
(54)
9
1
3
2
(69)
(8)
(23)
(17)
1
0
0
9
(8)
(0)
(0)
(75)
3
3
1
4
(43)
(43)
(14)
(57)
1
0
0
3
(14)
(0)
(0)
(43)
1
9
9
9
(8)
(69)
(69)
(69)
9
2
0
0
(69)
(15)
(0)
(0)
6
10
5
5
(50)
(77)
(42)
(42)
4
3
1
1
(31)
(25)
(8)
(8)
3
5
4
4
(43)
(71)
(57)
(57)
3
1
0
0
(43)
(14)
(0)
(0)
0
2
1
(0)
(15)
(8)
2
0
0
(15)
(0)
(0)
0
0
0
(0)
(0)
(0)
0
0
1
(0)
(0)
(8)
0
2
0
(0)
(28)
(0)
1
0
0
(14)
(0)
(0)
Grade 0–1 a
Grade 1–2 a
Toxicity graded by NCI common toxicity criteria (version 2.0).
a
Two values of grading were given because different grades occurred during different cycles in the same patient.
with locally advanced or locoregionally recurrent disease who
were expected to have a more favorable prognosis than our
patients with distant metastases [30]. At the diagnosis of distant metastases the percentage of patients with one, two or
more negative predictive factors for survival above reported
in the exploratory study group was the same or slightly higher
than in controls. Despite this a much more prolonged clinical
benefit and median survival than in the 30 controls occurred.
Concerning the explanation for our data, the following findings can be considered. (1) In experimental studies antiestrogens exerted a cytostatic effect on estrogen dependent but
also on estrogen independent tumor cells [42,44]; host natural killer function mediated in part the anti-tumor effect of
tamoxifen [1]. (2) Beta-interferon induced receptor enhance-
ment might be involved in the potentiation of the antiestrogen activity [6,32,35]. (3) The association of tamoxifen with
beta interferon suppressed tumor growth [32] and induced
cellular death through apoptosis by thioredoxin reductase [19].
This drug combination prevented tumor growth in vivo by
inhibition of tumor angiogenesis [18]. (4) Low dose betainterferon increased the natural killer activity of peripheral
blood mononuclear cells of breast cancer patients [39]. (5) In
animals anti-tumor activity of interleukin-2 was potentiated
by concomitant administration of tamoxifen [17]. (6) The antitumor activity of recombinant interleukin-2 included activation and expansion of cytotoxic T lymphocytes and natural
killer cells and the secretion of secondary cytokines such
gamma-interferon [11,34]. (7) The activity of interleukin-
262
A. Nicolini, A. Carpi / Biomedicine & Pharmacotherapy 59 (2005) 253–263
2 might have been favored by the overexpression of Major
Histocompatibily Complex I antigens on the cell surface due
to previous beta-interferon administration [3]. (8) Constitutive genetic activation in cancer cells suppresses innate and
adaptive anti-tumor immunity of the patients [43]. A production by tumoral cells of immunosuppressive factors has been
hypothesized [34].
The following hypothesis can be formulated. Estrogen
dependent cancer cells accumulate in G0/G1 phases following initial antiestrogen therapy. Successively beta interferon
and interleukin-2 potentiate the anti-tumor action of tamoxifen on both estrogen dependent and independent cell type.
Among the hypotheses on the acquired tamoxifen resistance
one is that breast cancer cells themselves (autocrine mechanism) and/or through the surrounding stromal cells (paracrine mechanism) are responsible for an increased growth factor expression [20]. Both tumor growth stimulating activities
and immunosuppression of innate and adaptive immunity
might be importantly reduced by our combined therapy. The
addition of beta-interferon to tamoxifen did not significantly
prolong response to hormone therapy and overall survival of
metastatic breast cancer patients [6,32]. The principal new
element we introduced was low dose cyclic interleukin2 administration when most tumoral cells may be quiescent
with low production of growth factors associated with lower
level of immunosuppression in the patient. Therefore, interleukin-2 may have a more favorable opportunity for stimulating the immune system. The cyclic 4 weeks rest from
immuno-therapy could favor the functional recovery of the
stimulated immune system activities induced by betainterferon and interleukin-2.
As far as we know no clinical study has tested the usefulness of interleukin-2 in hormone-responsive metastatic breast
cancer patients. In metastatic melanoma, a tumor without
endocrine dependence, tamoxifen, interleukin-2 and interferon alpha were unsuccessfully added to antiblastics [33].
An important favorable aspect of the drug association proposed in this study is the good tolerability and compliance
shown by the patients.
However, this investigation is an explorative study and confirmatory randomized trials are needed.
In conclusion these data strongly suggest that immunotherapy plays an important role in the treatment of human
cancer. The proposed hormone-immunotherapy is substantially innocuous and appears clinically very effective in metastatic breast cancer.
Acknowledgements
The authors thank:
Michelassi C., B.S. and Rossi G., Ph.D., from Unit of Epidemiology and Biostatistics, Institute of Clinical Physiology,
CNR, Pisa for the technical assistance in the statistical analysis.
Antonaci F.C., Ph.D. and Caccamo M., Assistant Professor, from University of Illinois at Urbana-Champaign for the
careful revision of the manuscript.
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