Cytokine & Growth Factor Reviews 17 (2006) 325–337

www.elsevier.com/locate/cytogfr

Mini review

Cytokines in breast cancer

A. Nicolini a,*, A. Carpi b, G. Rossi c
a
Department of Internal Medicine, University of Pisa, Via Roma 67, 56126 Pisa, Italy
b
Department of Ageing and Reproduction, University of Pisa, Italy
c
Unit of Epidemiology and Biostatistics, Institute of Clinical Physiology, CNR., Pisa, Italy

Available online 22 August 2006

Abstract

In recent decades many advances have occurred in the understanding of the role of cytokines in breast cancer. New signalling pathways of
interleukin (IL)-1 family, IL-6, IL-11, IL-18, interferons (IFNs) and interferon regulatory factors 1 (IRF-1) and 2 (IRF-2) have been found
within tumour microenvironments and in metastatic sites. Some cytokines (IL-1, IL-6, IL-11, TGFb) stimulate while others (IL-12, IL-18,
IFNs) inhibit breast cancer proliferation and/or invasion. Similarly, high circulating levels of some cytokines seem to be favourable (soluble
IL-2R) while others are unfavourable (IL-1b, IL-6, IL-8, IL-10, IL-18, gp130) prognostic indicators. So far IL-2, IFNa, IFNb and
occasionally IFNg, IL-6, IL-12 have been the cytokines used for anti tumour treatment of advanced breast cancer either to induce or increase
hormone sensitivity and/or to stimulate cellular immunity. Disappointing results occurred in most trials; however, two long-term pilot studies
suggest that IL-2 and IFNb, when used appropriately can have a positive effect on clinical benefit and overall survival of patients with minimal
residual disease after chemotherapy or with disseminated disease controlled by conventional endocrine therapy.
# 2006 Elsevier Ltd. All rights reserved.

Keywords: Breast cancer; Cytokines; Prognosis; Treatment

1. Introduction specific or non-specific antitumor responses. Furthermore,

Cytokines are glycoproteins of low molecular weight,
which are rapidly synthesized and usually secreted by
different healthy and diseased cells (mainly mononuclear
phagocytes and activated T lymphocytes) mainly after
stimulation. They act on many different adjacent target cells
(pleiotropism) often in an additive, synergistic, or antag-
onistic manner. In multicellular organisms, cytokines are
intercellular mediators that regulate survival, growth,
differentiation, and the effector functions of cells [1].
Therefore, it is not surprising that cytokines significantly
affect the growth of tumours in vivo. On the other hand, they
are also produced by cancer cells and represent a network
with a large variety of molecularly and functionally different
members that may act as tumour growth-promoting or
inhibiting factors. As they affect the growth and function of
immunocompetent cells, they can activate or modulate
* Corresponding author. Tel.: +39 050 992141; fax: +39 050 553414.
E-mail address: a.nicolini@int.med.unipi.it (A. Nicolini).
because cytokines are mediators of the effector response
from innate and acquired cellular immunities [2], they are
probably involved in the mechanism from tumour cell
evasion of the immunosurveillance system.
This review summarizes principal recent findings on the
relationship between cytokines and breast cancer and their
role in effecting patient prognosis. Additionally, this review
deeply explores the body of knowledge acquired in the last
decade for therapeutic antitumoral purpose in advanced
breast cancer.
2. Biological studies
2.1. Interleukins
2.1.1. Interactions with breast cancer cells
The interleukin (IL) -1 family of cytokines (IL-la, IL-
1b), the IL-1 receptor antagonist (IL-1Ra) and receptors (IL-
1RI and IL-1RII) have been found to be frequently expressed

1359-6101/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cytogfr.2006.07.002
326 A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337
in breast cancer cell lines, in human breast cancer tissue, and
within the tumour microenvironment [3–5]. This local
expression of IL-1/IL-1R cytokine family can control, via
autocrine and/or paracrine mechanisms, the tumour cell
subpopulation expression of other protumorigenic cyto-
kines, such as the expression of IL-8, and subsequently
contribute to protumorigenic activities, i.e. angiogenesis,
tumour proliferation, and local tumour invasion [4,5]. It has
been reported that IL-1b signalling pathway may be
different in ER positive MCF-7 versus ER negative
MDA-MB231 breast carcinoma cells [6].
IL-6 has been found in high concentrations in human breast
cancer cell lines and in breast tumour samples [7–9]. Fibro-
blasts, macrophages and lymphocytes (mainly Th2 cells) are
thought to be an important source of IL-6. This cytokine exerts
its effects through glycoprotein (gp) 130-mediated activation
of signalling pathways (including the JAK/STAT and MAP
kinase pathways) resulting in the transcriptional regulation of
genes involved in cell proliferation, survival and differentia-
tion. IL-6 promotes tumour growth by upregulating anti-
apoptotic and angiogenic proteins in tumour cells [1,10–12].
Increased IL-6 production also increases estradiol-17b
hydroxysteroid dehydrogenase (17b-HSD) type I which con-
verts estrone (E1) to the biologically active estrogen, estradiol
(E2) [13]. Therefore, it has been hypothesized that IL-6 and
IL-1b stimulate proliferation of breast cancer cells through
estrogen production by activating steroid-catalyzing enzymes
in the tissue [7]. IL-6 may favour proliferation and metastasis
of cancer cells, development of osteolysis and humoral
hypercalcemia, and it has also been suggested to be a
cachectic factor in cancer patients [8].
Bone metastases from breast cancer induce osteoclast
formation by stimulating osteoblastic production of IL-11
with the subsequent release of prostaglandin E2 and
inhibition of GM-CSF production by cells within the bone
microenvironment [14]. This mechanism seems to impor-
tantly contribute to breast cancer cell induced osteoclast
formation and their resorptive activity.
IL-12 is a heterodimeric molecule composed of an a
chain (p 35 subunit) and a b chain (p 40 subunit) linked to
form the biologically active 74 kDa heterodimer [15]. The
principal sources of IL-12 are macrophages, dendritic cells,
monocytes, neutrophils, and to a lesser extent, B cells.
Concomitant gene expression for IL-12 and interferon g was
demonstrated by reverse transcriptase-polymerase chain
reaction in all 10 cases of infiltrating ductal carcinoma [16].
IL-18 injected intraperitoneally, not only inhibited
osteolytic growth at bone metastatic sites of human breast
cancer cells, MDA-231 cells, in nude mice, but also
suppressed an early stage of bone metastasis. However, no
significant effect on proliferation of subcutaneously injected
cancer cells was found [17].
2.1.2. Prognostic role
In human breast cancer cells, IL-la protein expression
correlated with both poor differentiation and decreasing
epithelial ERa expression [3,5]. Elevated serum levels of IL-
1b correlated with a high rate of recurrence in patients with
breast cancer [18].
An association of prognostically favourable factors with
higher serum values of soluble IL-2 receptors has been
reported [19].
In serum of breast cancer patients, IL-6 correlated directly,
although not always [10], with clinical stage [20,21] and the
rate of recurrence [18]. In a few studies on metastatic breast
cancer, multivariate analysis identified high serum IL-6 levels
as an independent adverse prognostic variable for progression
free and overall survival [22–25]. Accordingly, the finding of
low serum IL-6 levels prior to and the maintenance of
relatively low IL-6 levels after 4 weeks of medroxyproges-
terone acetate treatment, was beneficial in 65 patients with
advanced or recurrent breast cancer [26].
Controversial findings have been reported with regard to
the relationship between IL-6 expression [27,28] or the
polymorphism at nucleotide-174 within the promoter region
of IL-6 gene [29,30] and clinical outcome.
Serum IL-8 levels were found higher in 69 patients with
either operable or advanced breast cancer as compared to
healthy women and they correlated directly with clinical stage
of breast cancer. Elevated serum IL-8 levels were also
associated with the occult cytokeratin-positive bone marrow
cells [31], thus suggesting a correlation with poor prognosis
[20,31]. Another study with 43 breast cancer patients
suggested that tissue and plasma b chemokine RANTES (a
member of the same family as IL-8) plays a role in
carcinogenesis and that a RANTES assay in tissue surround-
ing a tumour or in the tumour area after excision, may help to
predict unfavourable prognosis in breast cancer patients [32].
IL-10 levels in blood samples of breast cancer patients
were [20] or were not [32] higher than controls and
correlated directly with clinical stage of the disease [20].
Serum IL-18 levels were higher in breast cancer patients
than in control subjects, higher in advanced than in early
stages of the disease, and higher in metastatic than in non-
metastatic patients [33,34].
2.1.3. Effects on cellular immunity
This section reports the effects on cellular immunity of
the main interleukins used in clinical trials. IL-2 was purified
from mitogen-stimulated lymphocyte cultures and, in vivo,
is produced exclusively by activated T cells [35,36]. IL-2
induces the proliferation of activated T cells and the
differentiation of cytotoxic T lymphocytes (CTL); it also has
effects on other immune cells including natural killer (NK)
cells, B cells, monocyte/macrophages, and neutrophils [35–
37]. Three different IL-2 receptor (R) complexes exist; they
include three receptor subunits located on T and NK cells.
The isolated IL-2Ra binds IL-2 with low-affinity (Kd 
108 M) without transducing a signal; the heterodimeric IL-
2Rb g binds IL-2 with intermediate affinity (Kd  109 M)
and transduces intracellular signals while the heterotrimeric
IL-2Ra b g binds IL-2 with high affinity (Kd  1011 M)
 A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337
and also signals. The IL-2Rg also is referred to as the
common g chain (gc). The intermediate affinity receptor IL-
2Rbgc is expressed in most (90%) human NK cells that
are CD56dim NK cells. CD56dim NK cells have low surface
density expression of CD56 and high expression of CD16 and
NK receptors (NKRs). NKRs recognise MHC class I ligands
and regulate (inhibit or activate) the functional response to
target cells. CD56dim NK cells are potent mediators of
antibody-dependent cellular cytotoxicity (ADCC), lympho-
kine-activated killing (LAK) activity and natural cytotoxicity.
They do not express L-selectin and produce a low amount of
cytokines (IFNg, TNFa, TNFb3, IL-10) in response to
monokine stimulation. CD56bright NK cells are 10% of
human NK cells and express the high affinity IL-2Rabgc.
CD56bright NK cells have high-density expression of CD56,
low-density expression of CD16 and NKRs. They are poorly
cytotoxic but show potent LAK activity following activation
with IL-2; they highly express the adhesion molecule L-
selectin and through cytokine receptors produce elevated
levels of cytokine in response to monokine stimulation [37].
In cancer patients, CD56bright, unlike CD56dim, NK cells
proliferate in response to subcutaneously administered low or
ultralow (1 million IU/m2, pM serum concentration) doses
of IL-2 that selectively saturate and signal through IL2Rabgc.
Due to the constitutively high expression of L-selectin, the
expansion of CD56bright NK cells may result in an increased
number of NK cells capable of trafficking to secondary
lymphoid organs where they could interact with other immune
cells as T cells and antigen-presenting cells [37]. Higher doses
(720,000 IU/kg/q, nM serum concentration) of IL-2, admi-
nistered by continuous infusion or bolus, further stimulated
NK cells, activated T cells, monocyte/macrophages, and
activated B cells, which express the intermediate-affinity IL-
2Rbgc. These high IL-2 doses increase the toxicity by
CD56bright and CD56dim NK cells as well as their LAK
activity and ADCC, so that severe side effects can occur
(arterial hypotension, capillary leak syndrome). In an effort to
provide less toxic regimens, a different schedule of IL-2 using
an intermediate dose (72,000 IU/kg/q) was drawn. However,
this different schedule still resulted in relatively high (nM)
serum concentrations and was toxic [37].
Regarding IL-6 antitumour activity, induction of T cell
and B cell differentiation, stimulation of cytotoxic T cells
and help in producing lymphokine-activated killer cells have
been reported. Also, through the increased synthesis of C-
reactive protein (CRP) IL-6 indirectly influenced the binding
of this protein to phospholipids on tumour cells, activating
C1q of the complement system, which may lead to tumour
cell lysis [11,12].
The above mentioned study [16] indicated that breast
cancer induces a local IL-12-dependent type I immune
response likely directed towards tumour-associated anti-
gens. The major actions of IL-12 are on T and NK cells that
primarily express IL-12 receptors (IL-12Rbl and IL-12Rb2)
[38]. IL-12 is the main cytokine that regulates differentiation
of CD4+ T cells to the Th1 phenotype that produces IFNg
327
and promotes cell-mediated immunity. Furthermore, IL-12,
by inducing the Th1 response, also augments the production
of antibody classes able to activate the complement cascade
and to opsonise tumour cells exposing them to the cytotoxic
activity of phagocytic and NK cells.
IL-12 induces proliferation and increases cytotoxic
activity of CTLs and NK cells. Moreover, in these cells
through IL-12Rbl and IL-12Rb2, it activates the JAK/STAT
pathway and induces high production of IFNg which, in
turn, stimulates further IL-12 production by immunocom-
petent cells. IFNg, TNF, and a cascade of secondary and
tertiary proinflammatory cytokines induced by IL-12 also
stimulated the production of CXCR3 ligands, which are
powerful inhibitors of tumour angiogenesis [15,38].
Synergistic actions of IL-12 with IL-2 have been shown.
Low (pM) IL-2 doses can synergise with IL-12 for more
efficient CD56bright NK cell IFNg production [37] and IL-
12/IL-2 combination synergistically enhanced Fas and FasL
expression within tumors via an IFNg-dependent mechan-
ism in vivo. This combination also inhibited tumour neo-
vascularisation and induced rapid destruction of tumour-
associated endothelial cells and tumour regression [39].
2.2. Interferons
2.2.1. Interactions with breast cancer cells
a and b interferons are type I IFN proteins with antitumor
activity [2]. They downregulate oncogene expression and
induce tumour suppressor genes which result in antiproli-
ferative activity. Antiproliferative and antiadhesive actions
of IFNa have been shown in MCF-7 breast carcinoma cells
[40]. The antiproliferative effect of IFNa 2a and 2b on the
growth of ZR-75-1 human breast cancer cells was
synergistic with that of the antiestrogen, toremifene [41].
In human breast cancer, IFNa2a, combined with all-
transretinoic acid (AT), did not potentiate the growth
inhibition of AT [42]. With regard to hormone receptor
modulation by IFNa, conflicting results have been reported
[43–45].
Several genes associated with retinoid-IFN-induced
mortality (GRIM) have been recently identified. GRIM-12
expression was induced by IFNb/tamoxifen association at a
post-transcriptional stage [46] and overexpression of GRIM-
12 increased IFN/tamoxifen induced apoptosis. In two
human breast cancer cell lines (the ER-positive MCF-7 and
ER-negative MDA-MB-231 cells), the effects of huanglian
extract (a widely used herb in traditional Chinese medicine
with anticancer activities) on the expression of the common
genes involved in carcinogenesis were examined. In MCF-7
cells, the huanglian extract provoked a dramatic increase in
mRNA expression of IFNb and TNFa [47]. Estrogen and
progesterone receptor enhancement has been observed in
vitro and in patients affected by advanced breast cancer
treated with relatively low doses of IFNb [43].
IFNg, also called immune IFN or type II IFN, is
mainly produced by CD4+ Th1, CD8+, and NK cells [2]. In
328 A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337
cancer xenografts, the antiproliferative action of IFNg,
probably due to enhanced cell death by up regulation of
some caspases [48–50] and an antiangiogenic activity, have
been found [51].
In particular, many studies have been conducted on
interferon regulatory factors 1 (IRF-1) and 2 (IRF-2) that are
transcription factors in the interferon g signal transduction
pathway. IRF-1 acts as the effector arm of the interferon y
response with tumour suppressor activity. IRF-2 binds to the
same DNA consensus sequence and opposes IRF-1 activity
with oncogenic effects. High grade ductal carcinoma in situ
(DCIS) or node-positive invasive ductal cancers were found
less likely to express the tumour suppressor IRF-1 and much
more likely the oncogenic IRF-2 protein than normal tissue
[52]. In another study [48] on 187 specimens of clinically
defined invasive breast carcinoma, a significant positive
correlation between IRF-1 and IRF-2 expression and a
negative correlation between IRF-1 expression and tumour
grade were found. It has been hypothesised that IRF-1 acts as
a tumour suppressor gene and induces apoptosis [48-50].
Loss of IRF-1 regulation also appears related to antiestrogen
resistance. Antiestrogens induce both cytostasis (cell cycle
arrest) and apoptosis. In some breast cancer cell lines, the
lack of IRF-1 inhibited the ICI 182,780 (fulvestrant) induced
apoptotic response and reduced cell sensitivity to the
antiproliferative effects of this drug [53]. Other studies
[54,55] dealt with 3,30 -diindolylmethane (DIM) a natural
autolytic product in plants of the brassica genus and a
promising anticancer agent. DIM induced a G1 cell-cycle
arrest and strongly stimulated the cell cycle inhibitor p21
expression and promoter activity in both human estrogen
responsive and estrogen independent breast cancer cell lines
[54]. These studies showed that DIM can up regulate the
expression and stimulate the secretion of IFN-g in the
human MCF-7 breast cancer cell line [54,55]. In a further
report indole-3-carbinol (I3C), another naturally occurring
compound of brassica vegetables with anticancer properties,
has been investigated in MCF-7 human breast cancer cells
[56]. I3C mediated its anticancer effects also by stimulating
transcription of the IFNgRl gene and increasing the IFNg
response [56].
In human MCF-7 breast cancer cells, IFNg and AT acted
synergistically [57,58] to induce expression of FasR mRNA
and FasR protein which promote tumour cellular apoptosis.
IFNg signalling through IFNg receptor also activates signal
transducer and activator of transcription-1 (STAT1) protein
which belongs to the STAT family of transcription factors.
STAT1 and STAT3 members modulate IL-6 signalling
pathway and activate acute phase protein genes and a variety
of other genes [1]. In another study [59] on MCF-7 human
breast cancer cells, IFNg induced the overexpression of
retinoic acid inducible gene-I (RIG-I) which, in turn, up-
regulated the IFNg stimulated gene 15, able to amplify the
immunomodulatory effects of IFNg. The genotype analysis
of breast cancers showed that about 40% of the samples
examined [60] expressed the ‘‘interferon-related’’ signature
providing molecular support for a role for either inflamma-
tion or viral infection in the pathogenesis of breast cancer. In
other reports [61,62], IFNg polymorphism has been found in
association with an increased risk of sporadic breast cancer
development, probably related to a resultant compromised
immune surveillance.
No influence of hormone receptor status on the anti-
proliferative effects of IFNg in breast carcinoma cells in vitro
occurred [63] A synergistic inhibitory effect of IFNsg and a
was obtained in human breast cancer xenografts; moreover,
lesser responses occurred if they were given systemically
rather than directly into the tumour [64,65]. Finally, in human
tumour models, IFNs increased the apoptotic effect of 5-
fluorouracil [66,67], and the cyclophosphamide, paclitaxel
and doxorubicin cytotoxicity [67,68].
2.2.2. Prognostic role
In two studies on breast cancer patients, plasma IFNg
[32] and IFNg genotype [28] were not correlated with
disease course. In another study on 87 metastatic breast
cancer patients, decreased survival was significantly
associated with IFNg gene polymorphism (C-A repeats
within the first intron) and a higher IFNg transcription [69].
2.2.3. Effects on cellular immunity
Alpha and b IFNs increase the expression of MHC class I
molecules in tumour cells which can enhance immune
recognition [66].
More recently, it has been shown that type I IFNs have
further important immunological effects. They promote
human Th1 type of immune response, stimulate proliferation
and prolong survival of human cytotoxic lymphocytes, induce
maturation, and improve function of dendritic cells [66].
Moreover, in clinical trials on melanoma and renal cell
carcinoma, IFNa has been shown to be able to increase NK
activity and T helper lymphocytes, as well as in-vitro T-cell
responses and tumour infiltrating lymphocytes [66]. Further-
more, IFNs have shown, in vitro and in vivo, anti-angiogenic
activity [51,70]. Other studies in breast cancer patients
reported that also low or intermediate doses of IFNb provoked
an enhancement of NK cell cytotoxicity [71,72]. The
capability of type I IFNs to promote the rapid differentiation
of human monocytes into mature dendritic cells has become
of particular interest after the identification of ‘‘natural IFN-
producing cells’’ as CD4+CD11c type II dendritic cells
precursors also defined as plasmacytoid dentritic cells [66].
In breast cancer patients, the inhibitory action of adjuvant
CMF chemotherapy on NK cells was antagonised by the
addition of low IFNb dose [71].
Further actions of IFNg are the following: (a) increased
expression of MHC class I and class II molecules; (b)
activation of monocytes and macrophages able to induce a
cytokine cascade (IL-1b, IL-6, TNFa and IL-8) and a
successive complete activation of various subsets of T or B
cells; (c) differentiation of CD4+ to Th1 cells and inhibition
of Th2 proliferation [2,73,74].
 A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337
2.3. Other cytokines and receptors (TGFb, TNFa,
gp130)
Mechanisms by which TGFb arrests cell cycle in G1
phase in the nontransformed epithelial cells and loses this
inhibiting property in human breast cancer have been
identified and reviewed [75]. Fibroblasts provide structural
and biochemical support for breast cancer (i.e. desmoplastic
reaction); this effect did not occur following administration
of antibodies against TNFa (or IL-11) [76].
Gene expression of TGFb1 and TNFa was found in nine,
and in a single instance, of 10 cases of infiltrating ductal
carcinoma, respectively [16]. Additionally, no differences
were found in TNFa-alleles and genotype frequencies
between breast cancer patients and control subjects [62].
Constitutive activation of the STAT3 gene in human breast
cancer cells inhibited tumour secretion of pro-inflammatory
cytokines and produced soluble factors accounting for
inhibited innate and acquired cellular immunity [77].
Interestingly, inhibition of cytokine receptor gp130 signalling
in breast cancer blocked constitutive activation of STAT3 and
inhibited, in vivo, malignancy growth. Besides, dominant
negative gp130 protein MDA-231 breast cancer cells had
markedly decreased engraftment, size, and number of
metastases compared with control cells [78].
In a study [28], TGFb1 low production genotypes
(TGFb1-10 CC) were associated with an increased risk of
disease relapse. gp130 is one of two functional receptor
subunits of IL-6. Similar to IL-6, expression of gp130
correlated with good prognosis for patients with breast
cancer [27]. Conversely, serum gp130 levels were found to
increase significantly at each progressive tumour stage
without or after chemo/radiotherapy [10].
Table 1 summarizes the principal positive or negative
biological actions on tumour growth and useful prognostic
Table 1
Main recent data on cytokines involved in growth and prognosis of human breast cancer
Cytokine (s, t)(Ref)
Type
t IL-1 (a and b) [3–5,7]
t IL-1a, s IL-1b [3,5,18]
Soluble IL-2R [19]
t IL-6 [7,8,13,27,28]
s IL-6, t IL-8, s IL-8, s IL-10 [18,20–25,31,32]
t IL-11 [14]
t IL-12 [16]
i.p. IL-18 [17]
s IL-18 [33,34]
t IFNa (2a, 2b), t IFNb, t IFNg [2,40,41,46–51]
t IFNg p [61,62,69]
t TGFb [75]
t TGFbp [28]
t gp130 [27,78]
s gp130 [10]
For the details, see text. Abbreviations: Ref = reference; s = serum; t = tissue; i.p. = intraperitoneal; p = polymorphism; tumour growth: + = favoured;
 = inhibited; prognostic role: + = favourable;  = unfavourable; i = indefinite; c = controversial.
329
information of cytokines in breast cancer. Studies with
controversial results of the same cytokines are also reported.
3. Clinical trials in advanced breast cancer patients
Many cytokines have shown a potent therapeutic
antitumour effect in preclinical models. However, transla-
tion to clinical practice is limited, and at present, IFNa and
IL-2 are the only cytokines approved for oncologic
indications [79].
Table 2 shows the rationale and the therapeutic schedule
of the cytokines that have been used for antitumour
treatment of advanced breast cancer.
3.1. IL-2 given as single agent or with other cytokines
and/or other drugs
The main characteristics of the clinical trials with IL-2
given alone or with other cytokines and/or other drugs in the
treatment of advanced breast cancer are shown in Table 3a.
Five [80–83,86] out of the seven trials with IL-2 alone [80–
84] or IL-2 plus melatonin [85,86] were pilot studies. Three
[81,83,86] of the five pilot studies, one phase I and one phase
II trials [84,85] enrolled different cancer histotypes,
including few patients (from four to eight) with often
heavily pretreated breast cancer. In these five studies, the
small sample size did not permit any reliable consideration.
In the two remaining pilot studies [80,82] enrolling a slightly
larger number of breast cancer patients, IL-2 treatment was
considered ineffective.
Subcutaneous IL-2 and IFNa were consecutively [87] or
simultaneously [88] given at a low dose in another pilot
study and in one phase II non-randomised trial including a
higher number of advanced breast cancer patients after
Tumour growth Prognostic role
Proliferation Invasion
+ +

+
+ + c

i + i
  i
 i

  i
+ + 
+ + i

+ + +

330 A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337

Table 2
Cytokines used for antitumoral treatment of advanced breast cancer patients: rationale and therapeutic schedule
Cytokine Rationale Common schedules of therapy
IL-2 Stimulation of 3–5 days i.v. bolus 720,000 IU/kg/q (high dose; nM s.c.)
cellular immunity 3–5 days i.v. bolus 72,000 IU/kg/q (intermediate dose; nM s.c.)
1 million IU/m2 per day for 8 weeks
subcutaneously (low/ultralow dose; pM s.c.)
Type I IFN (a, b) Tumour growth inhibition; 1–10 MIU/m2 every day or
stimulation of cellular immunity; three-times a week for 2–4 weeks
modulation of hormone receptors; subcutaneously or i.m. or i.l. administration
enhanced cellular immune
response to cytotoxic agents
IFNg Tumour growth inhibition; 1  106 IU i.l. injections or 25 mg
stimulation of cellular immunity subcutaneously, days 1–5 of 4 weeks cycles
IL-6 Stimulation of cellular immunity 1–10 (25–30 MTD) mg/kg/day
subcutaneously; 10–100 mg/kg/day c.i.
IL-12 Stimulation of cellular immunity 250–500 ng/kg/day i.v. bolus twice weekly or for 5 days
For details see text; abbreviations: s.c. = serum concentration; i.l. = intralesional.

failure of previous conventional treatment. In the pilot study,
high-risk breast cancer patients were successfully treated
with high-dose chemotherapy and a IL-2-IFNa combination
after transplantation of autologous hematopoietic stem cells
activated by IL-2. The main aim was attained in 43% of the
evaluable patients who developed autologous graft-versus-
host disease (GVHD) in the hope of a graft-versus-tumour
(GVT) effect contributing to a lower relapse rate. The results
demonstrated the feasibility and the moderate toxicity of this
regimen. The mean follow-up was short (13 months) and the

Table 3a
Main clinical trials with interleukin-2 (IL-2) given as single agent or with other cytokines and/or other drugs as antitumoral treatment of advanced breast cancer
Cytokine Ref. Patients Trial Disease Immunological Clinical outcome
enrolled (n) Type Follow-up (months) stage effects

IL-2 [80] 10 Pilot n.r. Advanced Yes Ineffective (MR 2, SD 4)

[81] 4 Pilot n.r. Advanced Yes Ineffective (PD)
[82] 21 Pilot 71 (Median) Advanced n.r. Ineffective (2 years PFS and
OS 19% and 33%, respectively)
[83] 2 Pilot 8+ (Median) Metastatic Yes SD 1 (14 months)
[84] 8 Phase I n.r. Metastatic Yes Ineffective (two incomplete local
tumour regressions)
IL-2 vs. IL-2 [85] 7 Phase II 18 (Median) Advanced Yes ORR 0% vs. 33%
and MLT randomised
IL-2 and MLT [86] 6 Pilot n.r. Advanced Yes ORR 17%
IL-2 and IFNa [87] 34 Pilot 13 (Mean) Locally Yes 2 year DFS and OS 68% and
advanced 75%, respectively
[88] 40 Phase II n.r. Advanced n.r. Ineffective (ORR 3%; median OS
non-randomised 8.9 months; 8% C.B. 24 months)
IL-2 and trastuzumab [89] *33 Phase I n.r. Metastatic Yes ORR 17%
IL-2 and G-CSF [90] 43 Phase I n.r. Advanced Yes n.r.
IL-2 and epirubicin [91] 100 Phase III n.r. Metastatic n.r. ORR 64% vs. 58% and
vs. epirubicin randomised TTP 12 months 33% vs. 12%
IL-2, IFNb, MLT and [93] 29 Pilot 59 (Mean) Metastatic Yes Median C.B. 38 months;
TAM or toremifene median OS 103 months;
85% C.B. 24 months
For details see text. Ref. = reference; n.r. = not reported; MLT = melatonin; IFN = interferon; G-CSF = granulocyte colony stimulating factor; TAM = tamox-
ifen; MR = minor response; SD = stable disease; PD = progressive disease; C.B. = clinical benefit = CR + PR + SD; ORR = overall response rate; TTP = time
to progression; DFS = disease free survival; PFS = progression free survival; OS = overall survival; *with HER-2+ (2 pts), HER-2++ (6 pts) or HER-2+++ (25 pts)
tumours; RP = retinyl palmitate; ** IFNb 2 MIU (group A) vs. 6 MIU (group B) i.m. three-times a week for 2 weeks; ***arm a: MEGACE 160 mg daily; arm b:
IFNb 0.25 MIU + IFNg25 mg s.c. + MEGACE (as in arm a); arm c: IFNb 1 MIU/m2 + RP 100,000 IU/day b.m. plus TAM 30 mg/day b.m.; **** with HER-2++
(7) or HER-2+++ (5) tumours.
 A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337 331

observed overall and disease-free survival rates were
comparable to those reported in literature [87]. In the phase
II non-randomised trial, 40 advanced breast cancer patients
were recruited between May 1991 and March 1995 by 25
institutions throughout the States and the results were
published in 2004. Immunological effects were not reported
and the IL-2 IFNa combination was defined as ineffective
[88].
IL-2 plus trastuzumab (Herceptin), a humanised anti-
HER-2 mAb, was evaluated in a phase I trial that enrolled
patients with different cancer histotypes. Most of them (33
out of 45) had metastatic breast cancer that overexpressed
HER-2. All enrolled patients were not suitable for or
previously failed effective standard therapy. Clinical
benefit (2 CR, 2 PR and 9 SD) occurred in 56% of the
evaluated patients; no correlation was found between
immunological effects and clinical response; response
duration reported for the only two complete responders was
4 and 33 months, respectively. Therefore, IL-2 did not
increase the activity of trastuzumab [89]. In 43 advanced
breast cancer patients, IL-2 and granulocyte-colony
stimulating factor (G-CSF) were subcutaneously adminis-
tered in combination to develop a GVHD and GVT effect
[90]; no clinical antitumor outcome was reported. In a
randomised study, 100 hormone refractory patients
received IL-2 and epirubicin or epirubicin alone [91].
Encouraging results were obtained in favour of the IL-2-
epirubicin arm; however, no immunological effect was
described and the short follow-up, with no data on survival,
rendered the findings inconclusive.

Table 3b
Main clinical trials with IFNa, IFNb, IL-6, IL-12 given as single agent or with other cytokines (except IL-2) and/or other drugs as antitumoral treatment of
advanced breast cancer
Cytokine Ref. Patients Trial Disease Immunological Clinical outcome
enrolled (n) stage effects
Type Follow-up
(months)
IFNa [44] 20 Pilot n.r. Advanced n.r. ORR 10%
IFNa and TAM [94] 13 Pilot n.r. Advanced n.r. ORR 15.4%; median PFS
4 months (0–26 range)
[95] 7 Pilot n.r. Advanced n.r. ORR 57% (duration of
response 1–8 months)
[96] 21 Phase II n.r. Metastatic n.r. (non-effective in overcoming
non-randomised TAM resistance) ORR 50%
IFNa, IFNg alone [97] 11 Pilot n.r. Metastatic Yes CR in 53% of cutaneous
or combined recurrences
IFNb [98] 45 Pilot n.r. Metastatic n.r. **Group A: ORR 42%
(median duration 31 weeks);
group B: ORR 36%
(median duration 19 weeks)
IFNb and TAM [99] 43 Pilot n.r. Advanced n.r. ORR 26% (median duration 6 months)
[100] 30 Pilot n.r. Advanced n.r. ORR 13% (median duration 8 months)
[101] 33 Pilot n.r. Metastatic n.r. ORR 27% (median duration 7 months)
IFNb, RP [43] 36 Phase II 66 Metastatic n.r. Median OS 32 months
and TAM non-randomised (Median)
[102] 85 Phase II 84 Advanced n.r. ORR 59%; median OS 28 months
non-randomised (Median) (from trial entry) and 38 months
(from first metastasis)
IFNb, IFNg [103] 19 Phase II 32 Metastatic Yes Median OS 36 months; ORR 39%
and TAM randomised (Mean) (TTP 18 months, median OS 39 months)
IFNb, IFNg, [104] 60 Yes ***Arm a: ORR 22%; arm b: ORR 30%;
RP and TAM arm c: see Ref. [103]
or MAP
IL-6 [105] 1 Phase I n.r. Advanced Yes No antitumor response
IL-12 and [106] ****12 Phase I n.r. Metastatic Yes Ineffective; ORR 8%
trastuzumab
For details see text. Ref. = reference; n.r. = not reported; MLT = melatonin; IFN = interferon; G-CSF = granulocyte colony stimulating factor; TAM = tamox-
ifen; MR = minor response; SD = stable disease; PD = progressive disease; C.B. = clinical benefit = CR + PR + SD; ORR = overall response rate; TTP = time
to progression; DFS = disease free survival; PFS = progression free survival; OS = overall survival; *with HER-2+ (2 pts), HER-2++ (6 pts) or HER-2+++ (25 pts)
tumours; RP = retinyl palmitate; ** IFNb 2 MIU (group A) vs. 6 MIU (group B) i.m. three-times a week for 2 weeks; ***arm a: MEGACE 160 mg daily; arm b:
IFNb 0.25 MIU + IFNg25 mg s.c. + MEGACE (as in arm a); arm c: IFNb 1 MIU/m2 + RP 100,000 IU/day b.m. plus TAM 30 mg/day b.m.; **** with HER-2++
(7) or HER-2+++ (5) tumours.
332 A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337
In one study [92,93], metastatic patients, with responsive
or stable disease during antiestrogen first line salvage
therapy, were recruited for immunotherapy with IL-2, IFNb
and melatonin and subjected to prolonged follow-up. From
1992 to 2003, accrual was relatively slow. Nevertheless, the
median duration of clinical benefit and median overall
survival from the beginning of antiestrogen first line salvage
therapy in 29 evaluated patients was 38 and 103 months,
respectively, i.e. about three-times more than in historical
controls treated only with antiestrogens [93].
In summary, these studies showed that in locally
advanced or metastatic breast cancer patients IL-2 given
at low dose, alone or in association with other cytokines and/
or other drugs in an outpatient setting, is a well-tolerated
drug [81–93]. The response rate in three studies including
more than 10 breast cancer patients was 3% [88], 17% [89],
or 64% [91] and its duration was reported only in a few
patients. Median overall survival was reported in two studies
[88,92,93]. In one study [88] it was 8.9 months, as expected
without cytokine treatment, while in the other study it was
much longer (103 months) [92,93]. Therefore, in all trials,
except the last one, no relevant improvement of clinical
outcome in the treated patients was obtained although
occasional immunological effects were recorded.
3.2. IFNa, IFNb, IFNg, IL-6, IL-12 given as single
agent or with other cytokines (except IL-2) and/or other
drugs
The main characteristics of the clinical trials with IFNa,
IFNb, IFNg, IL-6, and IL-12, given alone or with other
cytokines and/or other drugs in the treatment of advanced
breast cancer, are shown in Table 3b. In the first four trials
[44,94–96], three pilot studies and one phase II non-
randomised trial, although with different schedules, IFNa
was given alone [44] or with tamoxifen [94–96]. In all four
studies, immunological effects were not recorded. How-
ever, in all [44,94,96] but one of them [95], IFNa did not
overcome tamoxifen resistance nor ameliorate clinical
outcomes. In this study [95], limited favourable results
were reported; metastatic skin or soft tissue lesions showed
increased ER expression and in 57% of patients, a response
lasting 1–8 months occurred. Similarly, in a pilot study
IFNa and IFNg, given locally, alone or combined, was
effective with 53% of CR in the treatment of cutaneous
recurrences associated with a well documented enhance-
ment of intralesional cell-mediated immunological
response [97]. However, in these last two studies [95,97]
the accrual was very low (7 and 11 patients, respectively).
In four other pilot studies [98–101] IFNb was given before
and/or concomitant with tamoxifen. Most patients had
positive or unknown receptor status. In the first study [98]
on patients with multiple soft tissue biopsable metastatic
breast cancer, a significant increase in tissue hormone
receptors after IFNb treatment occurred. However, the
clinical response rate and duration of response were in the
range of those described in similar patients treated only
with tamoxifen. In two further pilot studies [99,100]
advanced breast cancer patients treated with and non-
responsive, initially or after clinical benefit, to tamoxifen
were recruited to an IFNb-tamoxifen combination trial. In
the two studies, the overall response rate was 26 and 13%,
and median duration of response was 6 and 8 months;
stabilisation of disease occurred in 44 and 37% of the
patients, respectively. However, these studies have not
shown any significant prolongation of the expected overall
survival. In the last pilot study [101], IFNb did not improve
the efficacy of tamoxifen in a population of unselected
metastatic patients who had received no prior palliative
hormone therapy, no adjuvant tamoxifen, or had terminated
it at least 12 months previously. In all the studies with
IFNb, immunological effects were not recorded. In two
phase II non-randomised trials, IFNb and retinyl palmitate
were administered concomitant with tamoxifen until
progression [43,102]. In the first study, most patients had
metastatic disease and this therapy was given as main-
tenance only to the responders to previous conventional
chemotherapy. In the entire study population, median
overall survival was 32 months, as expected without
maintenance. However, in the 11 complete responders to
prior chemotherapy, median overall survival reached the
relatively long duration of 66 months [43]. In the other trial,
the same schedule of therapy was administered to advanced
breast cancer patients, pretreated with hormones or with
chemotherapy, and with evidence of progressive disease. In
the entire population, the overall response rate was 59% and
median overall survival was 38 months [102].
In a phase II randomised trial conducted in metastatic
patients, estrogen positive patients, previously treated with
tamoxifen, received medroxyprogesterone only (arm a) or
medroxyprogesterone plus IFNb and IFNg (arm b) while
patients with negative estrogen receptors were treated with
IFNb, IFNg and tamoxifen (arm c). In a first report [103],
the results obtained in arm c displaying the best clinical
outcome were analysed separately. In arm c, an objective
overall response rate of 39% and 18-month median duration
of response were achieved. Another 28% of patients
demonstrated stable disease with a median duration of 10
months. Median overall survival in the entire group was 36
months. These results are comparable to those commonly
observed in estrogen receptor positive patients. In the
subsequent report [104], changes in cytokine production in
the three groups of patients (arms a, b and c) were examined
and a correlation between serum IFNg increase and a
relatively favourable clinical outcome was found.
IL-6 did not produce any antitumor response in 11
patients with different histotypes of refractory advanced
malignancies including one inflammatory breast cancer
[105]. Eventually, in the last study highlighted in Table 3b on
the effects of IL-12 and trastuzumab, increased circulating
levels of IFNg, TNFa, and antiangiogenic factors were
found in patients with clinical benefit. Nevertheless, in this
 A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337
phase I trial, IL-12 did not enhance clinical efficacy of
trastuzumab [106].
In conclusion, IFNs given in an outpatient setting, are
responsible for relatively limited side effects. The overall
response rate (from 8 to 59%) is not so different from that
usually seen in estrogen resistant and estrogen sensitive
metastatic patients, respectively. Median overall survival
was reported only in three different studies and ranged from
32 to 38 months. Median overall survival of 32 and 38
months shown in two [43,102] of these three studies is
compatible with the studied population, also including
patients with locally advanced disease [107,108], while
median overall survival of 36 months in the other trial [103]
is slightly higher than that usually expected in a similar
population of patients with distant metastases [109–111]. A
relatively prolonged survival was reported only in the
subgroup of 11 patients on maintenance therapy with IFNb,
retinyl palmitate, and tamoxifen following complete
remission due to chemotherapy.
With regard to IL-6 and IL-12, in spite of the reported
immunological effects, they did not show any independent
or synergistic clinical effect.
4. Discussion and conclusions
In recent decades, many experimental in vitro and in
vivo studies have advanced the comprehension of the role
of cytokines in oncology. Some cytokines (IL-1, IL-6, IL-
11, TGFb) stimulate while others (IL-12, IL-18, IFNs)
inhibit breast cancer proliferation and/or invasion. Simi-
larly, high circulating levels of some cytokines seem to be
favourable (soluble IL-2R) while others are unfavourable
(IL-1b, IL-6, IL-8, IL-10, IL-18, gp130) prognostic
indicators. However, IL-2 is a potent stimulator of cellular
immunity and, for this property, is the most selected
interleukin for clinical trials. Interferons inhibit breast
cancer proliferation and/or invasion and they also stimulate
cellular immunity. Moreover, IFNb, in vitro and in vivo, has
been reported to enhance estrogen and progesterone
receptors. Many clinical trials evaluated the pharmacoki-
netic characteristics and antitumor action of cytokines. So
far, significant results have been reported with IL-2 and
IFNa in advanced malignant melanoma and renal cancer
[112–114]. In advanced breast cancer treatment, IL-2,
IFNa, IFNb and occasionally IFNg, IL-6, and IL-12 have
been used. While stimulation of the immunological cellular
response was the principal aim of IL-2, IFNg, IL-6 or IL-12
treatment, IFNa and IFNb were given to induce or increase
hormone dependency and overcome tamoxifen resistance
(Tables 3a and 3b).
Median overall survival was observed in few trials and it
was not better, except for one study [93], than that found in
similar populations treated only with conventional therapy
(Tables 3a and 3b). Therefore, the number of therapeutic
studies with cytokines has decreased notably in recent
333
years. Results were different in the non-randomised trial
[43] on patients responsive to previous conventional
chemotherapy and receiving a maintenance treatment with
low dose IFNb, retinyl palmitate and tamoxifen until
relapse (Table 3b). In this series, the 11 patients with
minimal residual disease, due to complete response to
chemotherapy, showed median and 9-year survival rates of
66 months and 34%, respectively [43]. In a different series
of 263 metastatic breast cancer patients in complete
remission following combination chemotherapy, but not
followed by any maintenance treatment, 42-month median
survival and about 15% 9-year survival were observed
[115]. However, the small sample size and some locally
advanced breast cancer patients, may have contributed to
the better outcome of the 11 complete responders with
maintenance therapy [43]. Furthermore, in this trial [43],
no immunological assessment was made. In a different
long-term study [93], 29 consecutive breast cancer patients
with distant metastases underwent immunotherapy with
IFNb and IL-2. Mean follow-up was 59  37 months
(mean  S.D.). All patients were selected following
clinical benefit on first line salvage therapy with
antiestrogen. Unlike the previous study, where the
encouraging result was observed in patients with minimal
residual disease, all 29 patients at the beginning of
immunotherapy had disseminated metastases. Therapy
with cytokines significantly prolonged clinical benefit and
overall survival in comparison to only antiestrogen therapy
of historical control and literature data [92–93]. In fact, 38
months median clinical benefit, 103 months median overall
survival and about 30% 9-year survival from the diagnosis
of relapse were found, that is about three-times more than
expected. During clinical benefit, stimulation of cellular
immunity occurred after IL-2, consistent with previous
reports [83,86,116], while it did not occur during disease
progression [93]. Further recent immunological assess-
ment confirmed findings on cellular immunity and showed
a different definite cytokine pattern during clinical benefit
and at the progression of disease [Nicolini et al., data not
shown]. It was hypothesised that an immunological cellular
response stimulated by IL-2 administration and by a
provoked inflammatory cytokine cascade was responsible
for the significant prolongation of the initial clinical benefit
due to endocrine therapy [93] [Nicolini et al., data not
shown]. Although with prolonged follow-up, this study
suffers from being a non-randomised trial.
In conclusion, these limited favourable findings suggest
that maintenance therapy with appropriate cytokines could
significantly improve clinical outcomes of advanced breast
cancer patients with minimal residual disease after
chemotherapy or with disseminated disease controlled by
conventional antiestrogens. Therefore, in these patients, IL-
2 and IFNs should be used with suitable schedules in
prospective randomised trials to confirm the prolongation of
the clinical benefit and overall survival due to conventional
chemo- or endocrine therapy.
334 A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337
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Andrea Nicolini is Clinical Researcher at the
Department of Internal Medicine at Pisa Univer-
sity. He was winner of fellowships at the Eur-
opean School of Oncology and of a grant by
Cancer Prevention and Detection Journal. His
research over the past 15 years was mainly
devoted to the field of breast cancer, in particular
to the use of serum tumour markers in the
‘‘early’’ detection and treatment of metastatic
disease and to the function of cell mediated
immunity. Another area of research was the role of needle aspiration
techniques and the use of tissue tumour markers (galectin 3) in the
preoperative diagnosis of thyroid cancer. Dr. Nicolini has published about
200 original papers (including chapters of books), most of them in peer
reviewed Journals and many review articles. He is teacher of Internal
337
Medicine and Clinical Oncology for academic Institutes besides he is
scientific advisor of Biomedicine and Pharmacotherapy and regularly has
served as reviewer for many scientific Journals.
Angelo Carpi is Clinical Researcher at the Uni-
versity Hospital of Pisa, 1972–1986. Associate
Professor of Internal Medicine, Pisa University,
1984 to today. Vice-Director Postgraduate
School of Endocrinology, 1997 to 2003, Medical
Faculty, University of Pisa. About 200 scientific
publications in peer-reviewed journals. Principal
fields: thyroid and breast tumours, male inferti-
lity, vascular endothelium. Editor of Biomedicine
& Pharmacotherapy. Reviewer for International
Journals with impact factor mainly in the fields of internal medicine and
endocrinology. Identified as international ‘recognised authority’ by the
UCLA School of Los Angeles and the Faculty of Medicine of the Haifa
University. Recently involved in research on biomaterials, director of a unit
in a project of the CNR (National Research Council) on biomaterials.
Dr. Giuseppe Rossi obtained his B.Sc. degree
from the University of Pisa in 1978 and his PhD
(Medical Statistics) in 1983 from the University
of Pavia. He currently is a Senior Scientist at the
National Research Council (Unit of Epidemiol-
ogy and Biostatistics – Institute of Clinical Phy-
siology) and Professor of Statistics in Medicine at
the University of Pisa. His research is focused on
clinical and environmental epidemiology. Dr.
Rossi has published about 100 original papers
(including chapters of books), most of them in peer reviewed Journals. He
served as secretary and treasurer of the Italian Region of the International
Biometric Society.