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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,


because cytokines are mediators of the effector response
Cytokines are glycoproteins of low molecular weight, from innate and acquired cellular immunities [2], they are
which are rapidly synthesized and usually secreted by probably involved in the mechanism from tumour cell
different healthy and diseased cells (mainly mononuclear evasion of the immunosurveillance system.
phagocytes and activated T lymphocytes) mainly after This review summarizes principal recent findings on the
stimulation. They act on many different adjacent target cells relationship between cytokines and breast cancer and their
(pleiotropism) often in an additive, synergistic, or antag- role in effecting patient prognosis. Additionally, this review
onistic manner. In multicellular organisms, cytokines are deeply explores the body of knowledge acquired in the last
intercellular mediators that regulate survival, growth, decade for therapeutic antitumoral purpose in advanced
differentiation, and the effector functions of cells [1]. breast cancer.
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 2. Biological studies
with a large variety of molecularly and functionally different
members that may act as tumour growth-promoting or 2.1. Interleukins
inhibiting factors. As they affect the growth and function of
immunocompetent cells, they can activate or modulate 2.1.1. Interactions with breast cancer cells
The interleukin (IL) -1 family of cytokines (IL-la, IL-
* Corresponding author. Tel.: +39 050 992141; fax: +39 050 553414. 1b), the IL-1 receptor antagonist (IL-1Ra) and receptors (IL-
E-mail address: a.nicolini@int.med.unipi.it (A. Nicolini). 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 epithelial ERa expression [3,5]. Elevated serum levels of IL-
within the tumour microenvironment [3–5]. This local 1b correlated with a high rate of recurrence in patients with
expression of IL-1/IL-1R cytokine family can control, via breast cancer [18].
autocrine and/or paracrine mechanisms, the tumour cell An association of prognostically favourable factors with
subpopulation expression of other protumorigenic cyto- higher serum values of soluble IL-2 receptors has been
kines, such as the expression of IL-8, and subsequently reported [19].
contribute to protumorigenic activities, i.e. angiogenesis, In serum of breast cancer patients, IL-6 correlated directly,
tumour proliferation, and local tumour invasion [4,5]. It has although not always [10], with clinical stage [20,21] and the
been reported that IL-1b signalling pathway may be rate of recurrence [18]. In a few studies on metastatic breast
different in ER positive MCF-7 versus ER negative cancer, multivariate analysis identified high serum IL-6 levels
MDA-MB231 breast carcinoma cells [6]. as an independent adverse prognostic variable for progression
IL-6 has been found in high concentrations in human breast free and overall survival [22–25]. Accordingly, the finding of
cancer cell lines and in breast tumour samples [7–9]. Fibro- low serum IL-6 levels prior to and the maintenance of
blasts, macrophages and lymphocytes (mainly Th2 cells) are relatively low IL-6 levels after 4 weeks of medroxyproges-
thought to be an important source of IL-6. This cytokine exerts terone acetate treatment, was beneficial in 65 patients with
its effects through glycoprotein (gp) 130-mediated activation advanced or recurrent breast cancer [26].
of signalling pathways (including the JAK/STAT and MAP Controversial findings have been reported with regard to
kinase pathways) resulting in the transcriptional regulation of the relationship between IL-6 expression [27,28] or the
genes involved in cell proliferation, survival and differentia- polymorphism at nucleotide-174 within the promoter region
tion. IL-6 promotes tumour growth by upregulating anti- of IL-6 gene [29,30] and clinical outcome.
apoptotic and angiogenic proteins in tumour cells [1,10–12]. Serum IL-8 levels were found higher in 69 patients with
Increased IL-6 production also increases estradiol-17b either operable or advanced breast cancer as compared to
hydroxysteroid dehydrogenase (17b-HSD) type I which con- healthy women and they correlated directly with clinical stage
verts estrone (E1) to the biologically active estrogen, estradiol of breast cancer. Elevated serum IL-8 levels were also
(E2) [13]. Therefore, it has been hypothesized that IL-6 and associated with the occult cytokeratin-positive bone marrow
IL-1b stimulate proliferation of breast cancer cells through cells [31], thus suggesting a correlation with poor prognosis
estrogen production by activating steroid-catalyzing enzymes [20,31]. Another study with 43 breast cancer patients
in the tissue [7]. IL-6 may favour proliferation and metastasis suggested that tissue and plasma b chemokine RANTES (a
of cancer cells, development of osteolysis and humoral member of the same family as IL-8) plays a role in
hypercalcemia, and it has also been suggested to be a carcinogenesis and that a RANTES assay in tissue surround-
cachectic factor in cancer patients [8]. ing a tumour or in the tumour area after excision, may help to
Bone metastases from breast cancer induce osteoclast predict unfavourable prognosis in breast cancer patients [32].
formation by stimulating osteoblastic production of IL-11 IL-10 levels in blood samples of breast cancer patients
with the subsequent release of prostaglandin E2 and were [20] or were not [32] higher than controls and
inhibition of GM-CSF production by cells within the bone correlated directly with clinical stage of the disease [20].
microenvironment [14]. This mechanism seems to impor- Serum IL-18 levels were higher in breast cancer patients
tantly contribute to breast cancer cell induced osteoclast than in control subjects, higher in advanced than in early
formation and their resorptive activity. stages of the disease, and higher in metastatic than in non-
IL-12 is a heterodimeric molecule composed of an a metastatic patients [33,34].
chain (p 35 subunit) and a b chain (p 40 subunit) linked to
form the biologically active 74 kDa heterodimer [15]. The 2.1.3. Effects on cellular immunity
principal sources of IL-12 are macrophages, dendritic cells, This section reports the effects on cellular immunity of
monocytes, neutrophils, and to a lesser extent, B cells. the main interleukins used in clinical trials. IL-2 was purified
Concomitant gene expression for IL-12 and interferon g was from mitogen-stimulated lymphocyte cultures and, in vivo,
demonstrated by reverse transcriptase-polymerase chain is produced exclusively by activated T cells [35,36]. IL-2
reaction in all 10 cases of infiltrating ductal carcinoma [16]. induces the proliferation of activated T cells and the
IL-18 injected intraperitoneally, not only inhibited differentiation of cytotoxic T lymphocytes (CTL); it also has
osteolytic growth at bone metastatic sites of human breast effects on other immune cells including natural killer (NK)
cancer cells, MDA-231 cells, in nude mice, but also cells, B cells, monocyte/macrophages, and neutrophils [35–
suppressed an early stage of bone metastasis. However, no 37]. Three different IL-2 receptor (R) complexes exist; they
significant effect on proliferation of subcutaneously injected include three receptor subunits located on T and NK cells.
cancer cells was found [17]. The isolated IL-2Ra binds IL-2 with low-affinity (Kd 
108 M) without transducing a signal; the heterodimeric IL-
2.1.2. Prognostic role 2Rb g binds IL-2 with intermediate affinity (Kd  109 M)
In human breast cancer cells, IL-la protein expression and transduces intracellular signals while the heterotrimeric
correlated with both poor differentiation and decreasing IL-2Ra b g binds IL-2 with high affinity (Kd  1011 M)
A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337 327

and also signals. The IL-2Rg also is referred to as the and promotes cell-mediated immunity. Furthermore, IL-12,
common g chain (gc). The intermediate affinity receptor IL- by inducing the Th1 response, also augments the production
2Rbgc is expressed in most (90%) human NK cells that of antibody classes able to activate the complement cascade
are CD56dim NK cells. CD56dim NK cells have low surface and to opsonise tumour cells exposing them to the cytotoxic
density expression of CD56 and high expression of CD16 and activity of phagocytic and NK cells.
NK receptors (NKRs). NKRs recognise MHC class I ligands IL-12 induces proliferation and increases cytotoxic
and regulate (inhibit or activate) the functional response to activity of CTLs and NK cells. Moreover, in these cells
target cells. CD56dim NK cells are potent mediators of through IL-12Rbl and IL-12Rb2, it activates the JAK/STAT
antibody-dependent cellular cytotoxicity (ADCC), lympho- pathway and induces high production of IFNg which, in
kine-activated killing (LAK) activity and natural cytotoxicity. turn, stimulates further IL-12 production by immunocom-
They do not express L-selectin and produce a low amount of petent cells. IFNg, TNF, and a cascade of secondary and
cytokines (IFNg, TNFa, TNFb3, IL-10) in response to tertiary proinflammatory cytokines induced by IL-12 also
monokine stimulation. CD56bright NK cells are 10% of stimulated the production of CXCR3 ligands, which are
human NK cells and express the high affinity IL-2Rabgc. powerful inhibitors of tumour angiogenesis [15,38].
CD56bright NK cells have high-density expression of CD56, Synergistic actions of IL-12 with IL-2 have been shown.
low-density expression of CD16 and NKRs. They are poorly Low (pM) IL-2 doses can synergise with IL-12 for more
cytotoxic but show potent LAK activity following activation efficient CD56bright NK cell IFNg production [37] and IL-
with IL-2; they highly express the adhesion molecule L- 12/IL-2 combination synergistically enhanced Fas and FasL
selectin and through cytokine receptors produce elevated expression within tumors via an IFNg-dependent mechan-
levels of cytokine in response to monokine stimulation [37]. ism in vivo. This combination also inhibited tumour neo-
In cancer patients, CD56bright, unlike CD56dim, NK cells vascularisation and induced rapid destruction of tumour-
proliferate in response to subcutaneously administered low or associated endothelial cells and tumour regression [39].
ultralow (1 million IU/m2, pM serum concentration) doses
of IL-2 that selectively saturate and signal through IL2Rabgc. 2.2. Interferons
Due to the constitutively high expression of L-selectin, the
expansion of CD56bright NK cells may result in an increased 2.2.1. Interactions with breast cancer cells
number of NK cells capable of trafficking to secondary a and b interferons are type I IFN proteins with antitumor
lymphoid organs where they could interact with other immune activity [2]. They downregulate oncogene expression and
cells as T cells and antigen-presenting cells [37]. Higher doses induce tumour suppressor genes which result in antiproli-
(720,000 IU/kg/q, nM serum concentration) of IL-2, admi- ferative activity. Antiproliferative and antiadhesive actions
nistered by continuous infusion or bolus, further stimulated of IFNa have been shown in MCF-7 breast carcinoma cells
NK cells, activated T cells, monocyte/macrophages, and [40]. The antiproliferative effect of IFNa 2a and 2b on the
activated B cells, which express the intermediate-affinity IL- growth of ZR-75-1 human breast cancer cells was
2Rbgc. These high IL-2 doses increase the toxicity by synergistic with that of the antiestrogen, toremifene [41].
CD56bright and CD56dim NK cells as well as their LAK In human breast cancer, IFNa2a, combined with all-
activity and ADCC, so that severe side effects can occur transretinoic acid (AT), did not potentiate the growth
(arterial hypotension, capillary leak syndrome). In an effort to inhibition of AT [42]. With regard to hormone receptor
provide less toxic regimens, a different schedule of IL-2 using modulation by IFNa, conflicting results have been reported
an intermediate dose (72,000 IU/kg/q) was drawn. However, [43–45].
this different schedule still resulted in relatively high (nM) Several genes associated with retinoid-IFN-induced
serum concentrations and was toxic [37]. mortality (GRIM) have been recently identified. GRIM-12
Regarding IL-6 antitumour activity, induction of T cell expression was induced by IFNb/tamoxifen association at a
and B cell differentiation, stimulation of cytotoxic T cells post-transcriptional stage [46] and overexpression of GRIM-
and help in producing lymphokine-activated killer cells have 12 increased IFN/tamoxifen induced apoptosis. In two
been reported. Also, through the increased synthesis of C- human breast cancer cell lines (the ER-positive MCF-7 and
reactive protein (CRP) IL-6 indirectly influenced the binding ER-negative MDA-MB-231 cells), the effects of huanglian
of this protein to phospholipids on tumour cells, activating extract (a widely used herb in traditional Chinese medicine
C1q of the complement system, which may lead to tumour with anticancer activities) on the expression of the common
cell lysis [11,12]. genes involved in carcinogenesis were examined. In MCF-7
The above mentioned study [16] indicated that breast cells, the huanglian extract provoked a dramatic increase in
cancer induces a local IL-12-dependent type I immune mRNA expression of IFNb and TNFa [47]. Estrogen and
response likely directed towards tumour-associated anti- progesterone receptor enhancement has been observed in
gens. The major actions of IL-12 are on T and NK cells that vitro and in patients affected by advanced breast cancer
primarily express IL-12 receptors (IL-12Rbl and IL-12Rb2) treated with relatively low doses of IFNb [43].
[38]. IL-12 is the main cytokine that regulates differentiation IFNg, also called immune IFN or type II IFN, is
of CD4+ T cells to the Th1 phenotype that produces IFNg 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, providing molecular support for a role for either inflamma-
probably due to enhanced cell death by up regulation of tion or viral infection in the pathogenesis of breast cancer. In
some caspases [48–50] and an antiangiogenic activity, have other reports [61,62], IFNg polymorphism has been found in
been found [51]. association with an increased risk of sporadic breast cancer
In particular, many studies have been conducted on development, probably related to a resultant compromised
interferon regulatory factors 1 (IRF-1) and 2 (IRF-2) that are immune surveillance.
transcription factors in the interferon g signal transduction No influence of hormone receptor status on the anti-
pathway. IRF-1 acts as the effector arm of the interferon y proliferative effects of IFNg in breast carcinoma cells in vitro
response with tumour suppressor activity. IRF-2 binds to the occurred [63] A synergistic inhibitory effect of IFNsg and a
same DNA consensus sequence and opposes IRF-1 activity was obtained in human breast cancer xenografts; moreover,
with oncogenic effects. High grade ductal carcinoma in situ lesser responses occurred if they were given systemically
(DCIS) or node-positive invasive ductal cancers were found rather than directly into the tumour [64,65]. Finally, in human
less likely to express the tumour suppressor IRF-1 and much tumour models, IFNs increased the apoptotic effect of 5-
more likely the oncogenic IRF-2 protein than normal tissue fluorouracil [66,67], and the cyclophosphamide, paclitaxel
[52]. In another study [48] on 187 specimens of clinically and doxorubicin cytotoxicity [67,68].
defined invasive breast carcinoma, a significant positive
correlation between IRF-1 and IRF-2 expression and a 2.2.2. Prognostic role
negative correlation between IRF-1 expression and tumour In two studies on breast cancer patients, plasma IFNg
grade were found. It has been hypothesised that IRF-1 acts as [32] and IFNg genotype [28] were not correlated with
a tumour suppressor gene and induces apoptosis [48-50]. disease course. In another study on 87 metastatic breast
Loss of IRF-1 regulation also appears related to antiestrogen cancer patients, decreased survival was significantly
resistance. Antiestrogens induce both cytostasis (cell cycle associated with IFNg gene polymorphism (C-A repeats
arrest) and apoptosis. In some breast cancer cell lines, the within the first intron) and a higher IFNg transcription [69].
lack of IRF-1 inhibited the ICI 182,780 (fulvestrant) induced
apoptotic response and reduced cell sensitivity to the 2.2.3. Effects on cellular immunity
antiproliferative effects of this drug [53]. Other studies Alpha and b IFNs increase the expression of MHC class I
[54,55] dealt with 3,30 -diindolylmethane (DIM) a natural molecules in tumour cells which can enhance immune
autolytic product in plants of the brassica genus and a recognition [66].
promising anticancer agent. DIM induced a G1 cell-cycle More recently, it has been shown that type I IFNs have
arrest and strongly stimulated the cell cycle inhibitor p21 further important immunological effects. They promote
expression and promoter activity in both human estrogen human Th1 type of immune response, stimulate proliferation
responsive and estrogen independent breast cancer cell lines and prolong survival of human cytotoxic lymphocytes, induce
[54]. These studies showed that DIM can up regulate the maturation, and improve function of dendritic cells [66].
expression and stimulate the secretion of IFN-g in the Moreover, in clinical trials on melanoma and renal cell
human MCF-7 breast cancer cell line [54,55]. In a further carcinoma, IFNa has been shown to be able to increase NK
report indole-3-carbinol (I3C), another naturally occurring activity and T helper lymphocytes, as well as in-vitro T-cell
compound of brassica vegetables with anticancer properties, responses and tumour infiltrating lymphocytes [66]. Further-
has been investigated in MCF-7 human breast cancer cells more, IFNs have shown, in vitro and in vivo, anti-angiogenic
[56]. I3C mediated its anticancer effects also by stimulating activity [51,70]. Other studies in breast cancer patients
transcription of the IFNgRl gene and increasing the IFNg reported that also low or intermediate doses of IFNb provoked
response [56]. an enhancement of NK cell cytotoxicity [71,72]. The
In human MCF-7 breast cancer cells, IFNg and AT acted capability of type I IFNs to promote the rapid differentiation
synergistically [57,58] to induce expression of FasR mRNA of human monocytes into mature dendritic cells has become
and FasR protein which promote tumour cellular apoptosis. of particular interest after the identification of ‘‘natural IFN-
IFNg signalling through IFNg receptor also activates signal producing cells’’ as CD4+CD11c type II dendritic cells
transducer and activator of transcription-1 (STAT1) protein precursors also defined as plasmacytoid dentritic cells [66].
which belongs to the STAT family of transcription factors. In breast cancer patients, the inhibitory action of adjuvant
STAT1 and STAT3 members modulate IL-6 signalling CMF chemotherapy on NK cells was antagonised by the
pathway and activate acute phase protein genes and a variety addition of low IFNb dose [71].
of other genes [1]. In another study [59] on MCF-7 human Further actions of IFNg are the following: (a) increased
breast cancer cells, IFNg induced the overexpression of expression of MHC class I and class II molecules; (b)
retinoic acid inducible gene-I (RIG-I) which, in turn, up- activation of monocytes and macrophages able to induce a
regulated the IFNg stimulated gene 15, able to amplify the cytokine cascade (IL-1b, IL-6, TNFa and IL-8) and a
immunomodulatory effects of IFNg. The genotype analysis successive complete activation of various subsets of T or B
of breast cancers showed that about 40% of the samples cells; (c) differentiation of CD4+ to Th1 cells and inhibition
examined [60] expressed the ‘‘interferon-related’’ signature of Th2 proliferation [2,73,74].
A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337 329

2.3. Other cytokines and receptors (TGFb, TNFa, information of cytokines in breast cancer. Studies with
gp130) controversial results of the same cytokines are also reported.

Mechanisms by which TGFb arrests cell cycle in G1


phase in the nontransformed epithelial cells and loses this 3. Clinical trials in advanced breast cancer patients
inhibiting property in human breast cancer have been
identified and reviewed [75]. Fibroblasts provide structural Many cytokines have shown a potent therapeutic
and biochemical support for breast cancer (i.e. desmoplastic antitumour effect in preclinical models. However, transla-
reaction); this effect did not occur following administration tion to clinical practice is limited, and at present, IFNa and
of antibodies against TNFa (or IL-11) [76]. IL-2 are the only cytokines approved for oncologic
Gene expression of TGFb1 and TNFa was found in nine, indications [79].
and in a single instance, of 10 cases of infiltrating ductal Table 2 shows the rationale and the therapeutic schedule
carcinoma, respectively [16]. Additionally, no differences of the cytokines that have been used for antitumour
were found in TNFa-alleles and genotype frequencies treatment of advanced breast cancer.
between breast cancer patients and control subjects [62].
Constitutive activation of the STAT3 gene in human breast 3.1. IL-2 given as single agent or with other cytokines
cancer cells inhibited tumour secretion of pro-inflammatory and/or other drugs
cytokines and produced soluble factors accounting for
inhibited innate and acquired cellular immunity [77]. The main characteristics of the clinical trials with IL-2
Interestingly, inhibition of cytokine receptor gp130 signalling given alone or with other cytokines and/or other drugs in the
in breast cancer blocked constitutive activation of STAT3 and treatment of advanced breast cancer are shown in Table 3a.
inhibited, in vivo, malignancy growth. Besides, dominant Five [80–83,86] out of the seven trials with IL-2 alone [80–
negative gp130 protein MDA-231 breast cancer cells had 84] or IL-2 plus melatonin [85,86] were pilot studies. Three
markedly decreased engraftment, size, and number of [81,83,86] of the five pilot studies, one phase I and one phase
metastases compared with control cells [78]. II trials [84,85] enrolled different cancer histotypes,
In a study [28], TGFb1 low production genotypes including few patients (from four to eight) with often
(TGFb1-10 CC) were associated with an increased risk of heavily pretreated breast cancer. In these five studies, the
disease relapse. gp130 is one of two functional receptor small sample size did not permit any reliable consideration.
subunits of IL-6. Similar to IL-6, expression of gp130 In the two remaining pilot studies [80,82] enrolling a slightly
correlated with good prognosis for patients with breast larger number of breast cancer patients, IL-2 treatment was
cancer [27]. Conversely, serum gp130 levels were found to considered ineffective.
increase significantly at each progressive tumour stage Subcutaneous IL-2 and IFNa were consecutively [87] or
without or after chemo/radiotherapy [10]. simultaneously [88] given at a low dose in another pilot
Table 1 summarizes the principal positive or negative study and in one phase II non-randomised trial including a
biological actions on tumour growth and useful prognostic higher number of advanced breast cancer patients after

Table 1
Main recent data on cytokines involved in growth and prognosis of human breast cancer
Cytokine (s, t)(Ref) Tumour growth Prognostic role
Type Proliferation Invasion
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] + + c
s IL-6, t IL-8, s IL-8, s IL-10 [18,20–25,31,32] 
t IL-11 [14] i + i
t IL-12 [16]   i
i.p. IL-18 [17]  i
s IL-18 [33,34] 
t IFNa (2a, 2b), t IFNb, t IFNg [2,40,41,46–51]   i
t IFNg p [61,62,69] + + 
t TGFb [75] + + i
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.
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, evaluable patients who developed autologous graft-versus-
high-risk breast cancer patients were successfully treated host disease (GVHD) in the hope of a graft-versus-tumour
with high-dose chemotherapy and a IL-2-IFNa combination (GVT) effect contributing to a lower relapse rate. The results
after transplantation of autologous hematopoietic stem cells demonstrated the feasibility and the moderate toxicity of this
activated by IL-2. The main aim was attained in 43% of the 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 evaluated patients; no correlation was found between
comparable to those reported in literature [87]. In the phase immunological effects and clinical response; response
II non-randomised trial, 40 advanced breast cancer patients duration reported for the only two complete responders was
were recruited between May 1991 and March 1995 by 25 4 and 33 months, respectively. Therefore, IL-2 did not
institutions throughout the States and the results were increase the activity of trastuzumab [89]. In 43 advanced
published in 2004. Immunological effects were not reported breast cancer patients, IL-2 and granulocyte-colony
and the IL-2 IFNa combination was defined as ineffective stimulating factor (G-CSF) were subcutaneously adminis-
[88]. tered in combination to develop a GVHD and GVT effect
IL-2 plus trastuzumab (Herceptin), a humanised anti- [90]; no clinical antitumor outcome was reported. In a
HER-2 mAb, was evaluated in a phase I trial that enrolled randomised study, 100 hormone refractory patients
patients with different cancer histotypes. Most of them (33 received IL-2 and epirubicin or epirubicin alone [91].
out of 45) had metastatic breast cancer that overexpressed Encouraging results were obtained in favour of the IL-2-
HER-2. All enrolled patients were not suitable for or epirubicin arm; however, no immunological effect was
previously failed effective standard therapy. Clinical described and the short follow-up, with no data on survival,
benefit (2 CR, 2 PR and 9 SD) occurred in 56% of the 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 range of those described in similar patients treated only
or stable disease during antiestrogen first line salvage with tamoxifen. In two further pilot studies [99,100]
therapy, were recruited for immunotherapy with IL-2, IFNb advanced breast cancer patients treated with and non-
and melatonin and subjected to prolonged follow-up. From responsive, initially or after clinical benefit, to tamoxifen
1992 to 2003, accrual was relatively slow. Nevertheless, the were recruited to an IFNb-tamoxifen combination trial. In
median duration of clinical benefit and median overall the two studies, the overall response rate was 26 and 13%,
survival from the beginning of antiestrogen first line salvage and median duration of response was 6 and 8 months;
therapy in 29 evaluated patients was 38 and 103 months, stabilisation of disease occurred in 44 and 37% of the
respectively, i.e. about three-times more than in historical patients, respectively. However, these studies have not
controls treated only with antiestrogens [93]. shown any significant prolongation of the expected overall
In summary, these studies showed that in locally survival. In the last pilot study [101], IFNb did not improve
advanced or metastatic breast cancer patients IL-2 given the efficacy of tamoxifen in a population of unselected
at low dose, alone or in association with other cytokines and/ metastatic patients who had received no prior palliative
or other drugs in an outpatient setting, is a well-tolerated hormone therapy, no adjuvant tamoxifen, or had terminated
drug [81–93]. The response rate in three studies including it at least 12 months previously. In all the studies with
more than 10 breast cancer patients was 3% [88], 17% [89], IFNb, immunological effects were not recorded. In two
or 64% [91] and its duration was reported only in a few phase II non-randomised trials, IFNb and retinyl palmitate
patients. Median overall survival was reported in two studies were administered concomitant with tamoxifen until
[88,92,93]. In one study [88] it was 8.9 months, as expected progression [43,102]. In the first study, most patients had
without cytokine treatment, while in the other study it was metastatic disease and this therapy was given as main-
much longer (103 months) [92,93]. Therefore, in all trials, tenance only to the responders to previous conventional
except the last one, no relevant improvement of clinical chemotherapy. In the entire study population, median
outcome in the treated patients was obtained although overall survival was 32 months, as expected without
occasional immunological effects were recorded. maintenance. However, in the 11 complete responders to
prior chemotherapy, median overall survival reached the
3.2. IFNa, IFNb, IFNg, IL-6, IL-12 given as single relatively long duration of 66 months [43]. In the other trial,
agent or with other cytokines (except IL-2) and/or other the same schedule of therapy was administered to advanced
drugs breast cancer patients, pretreated with hormones or with
chemotherapy, and with evidence of progressive disease. In
The main characteristics of the clinical trials with IFNa, the entire population, the overall response rate was 59% and
IFNb, IFNg, IL-6, and IL-12, given alone or with other median overall survival was 38 months [102].
cytokines and/or other drugs in the treatment of advanced In a phase II randomised trial conducted in metastatic
breast cancer, are shown in Table 3b. In the first four trials patients, estrogen positive patients, previously treated with
[44,94–96], three pilot studies and one phase II non- tamoxifen, received medroxyprogesterone only (arm a) or
randomised trial, although with different schedules, IFNa medroxyprogesterone plus IFNb and IFNg (arm b) while
was given alone [44] or with tamoxifen [94–96]. In all four patients with negative estrogen receptors were treated with
studies, immunological effects were not recorded. How- IFNb, IFNg and tamoxifen (arm c). In a first report [103],
ever, in all [44,94,96] but one of them [95], IFNa did not the results obtained in arm c displaying the best clinical
overcome tamoxifen resistance nor ameliorate clinical outcome were analysed separately. In arm c, an objective
outcomes. In this study [95], limited favourable results overall response rate of 39% and 18-month median duration
were reported; metastatic skin or soft tissue lesions showed of response were achieved. Another 28% of patients
increased ER expression and in 57% of patients, a response demonstrated stable disease with a median duration of 10
lasting 1–8 months occurred. Similarly, in a pilot study months. Median overall survival in the entire group was 36
IFNa and IFNg, given locally, alone or combined, was months. These results are comparable to those commonly
effective with 53% of CR in the treatment of cutaneous observed in estrogen receptor positive patients. In the
recurrences associated with a well documented enhance- subsequent report [104], changes in cytokine production in
ment of intralesional cell-mediated immunological the three groups of patients (arms a, b and c) were examined
response [97]. However, in these last two studies [95,97] and a correlation between serum IFNg increase and a
the accrual was very low (7 and 11 patients, respectively). relatively favourable clinical outcome was found.
In four other pilot studies [98–101] IFNb was given before IL-6 did not produce any antitumor response in 11
and/or concomitant with tamoxifen. Most patients had patients with different histotypes of refractory advanced
positive or unknown receptor status. In the first study [98] malignancies including one inflammatory breast cancer
on patients with multiple soft tissue biopsable metastatic [105]. Eventually, in the last study highlighted in Table 3b on
breast cancer, a significant increase in tissue hormone the effects of IL-12 and trastuzumab, increased circulating
receptors after IFNb treatment occurred. However, the levels of IFNg, TNFa, and antiangiogenic factors were
clinical response rate and duration of response were in the found in patients with clinical benefit. Nevertheless, in this
A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337 333

phase I trial, IL-12 did not enhance clinical efficacy of years. Results were different in the non-randomised trial
trastuzumab [106]. [43] on patients responsive to previous conventional
In conclusion, IFNs given in an outpatient setting, are chemotherapy and receiving a maintenance treatment with
responsible for relatively limited side effects. The overall low dose IFNb, retinyl palmitate and tamoxifen until
response rate (from 8 to 59%) is not so different from that relapse (Table 3b). In this series, the 11 patients with
usually seen in estrogen resistant and estrogen sensitive minimal residual disease, due to complete response to
metastatic patients, respectively. Median overall survival chemotherapy, showed median and 9-year survival rates of
was reported only in three different studies and ranged from 66 months and 34%, respectively [43]. In a different series
32 to 38 months. Median overall survival of 32 and 38 of 263 metastatic breast cancer patients in complete
months shown in two [43,102] of these three studies is remission following combination chemotherapy, but not
compatible with the studied population, also including followed by any maintenance treatment, 42-month median
patients with locally advanced disease [107,108], while survival and about 15% 9-year survival were observed
median overall survival of 36 months in the other trial [103] [115]. However, the small sample size and some locally
is slightly higher than that usually expected in a similar advanced breast cancer patients, may have contributed to
population of patients with distant metastases [109–111]. A the better outcome of the 11 complete responders with
relatively prolonged survival was reported only in the maintenance therapy [43]. Furthermore, in this trial [43],
subgroup of 11 patients on maintenance therapy with IFNb, no immunological assessment was made. In a different
retinyl palmitate, and tamoxifen following complete long-term study [93], 29 consecutive breast cancer patients
remission due to chemotherapy. with distant metastases underwent immunotherapy with
With regard to IL-6 and IL-12, in spite of the reported IFNb and IL-2. Mean follow-up was 59  37 months
immunological effects, they did not show any independent (mean  S.D.). All patients were selected following
or synergistic clinical effect. clinical benefit on first line salvage therapy with
antiestrogen. Unlike the previous study, where the
encouraging result was observed in patients with minimal
4. Discussion and conclusions residual disease, all 29 patients at the beginning of
immunotherapy had disseminated metastases. Therapy
In recent decades, many experimental in vitro and in with cytokines significantly prolonged clinical benefit and
vivo studies have advanced the comprehension of the role overall survival in comparison to only antiestrogen therapy
of cytokines in oncology. Some cytokines (IL-1, IL-6, IL- of historical control and literature data [92–93]. In fact, 38
11, TGFb) stimulate while others (IL-12, IL-18, IFNs) months median clinical benefit, 103 months median overall
inhibit breast cancer proliferation and/or invasion. Simi- survival and about 30% 9-year survival from the diagnosis
larly, high circulating levels of some cytokines seem to be of relapse were found, that is about three-times more than
favourable (soluble IL-2R) while others are unfavourable expected. During clinical benefit, stimulation of cellular
(IL-1b, IL-6, IL-8, IL-10, IL-18, gp130) prognostic immunity occurred after IL-2, consistent with previous
indicators. However, IL-2 is a potent stimulator of cellular reports [83,86,116], while it did not occur during disease
immunity and, for this property, is the most selected progression [93]. Further recent immunological assess-
interleukin for clinical trials. Interferons inhibit breast ment confirmed findings on cellular immunity and showed
cancer proliferation and/or invasion and they also stimulate a different definite cytokine pattern during clinical benefit
cellular immunity. Moreover, IFNb, in vitro and in vivo, has and at the progression of disease [Nicolini et al., data not
been reported to enhance estrogen and progesterone shown]. It was hypothesised that an immunological cellular
receptors. Many clinical trials evaluated the pharmacoki- response stimulated by IL-2 administration and by a
netic characteristics and antitumor action of cytokines. So provoked inflammatory cytokine cascade was responsible
far, significant results have been reported with IL-2 and for the significant prolongation of the initial clinical benefit
IFNa in advanced malignant melanoma and renal cancer due to endocrine therapy [93] [Nicolini et al., data not
[112–114]. In advanced breast cancer treatment, IL-2, shown]. Although with prolonged follow-up, this study
IFNa, IFNb and occasionally IFNg, IL-6, and IL-12 have suffers from being a non-randomised trial.
been used. While stimulation of the immunological cellular In conclusion, these limited favourable findings suggest
response was the principal aim of IL-2, IFNg, IL-6 or IL-12 that maintenance therapy with appropriate cytokines could
treatment, IFNa and IFNb were given to induce or increase significantly improve clinical outcomes of advanced breast
hormone dependency and overcome tamoxifen resistance cancer patients with minimal residual disease after
(Tables 3a and 3b). chemotherapy or with disseminated disease controlled by
Median overall survival was observed in few trials and it conventional antiestrogens. Therefore, in these patients, IL-
was not better, except for one study [93], than that found in 2 and IFNs should be used with suitable schedules in
similar populations treated only with conventional therapy prospective randomised trials to confirm the prolongation of
(Tables 3a and 3b). Therefore, the number of therapeutic the clinical benefit and overall survival due to conventional
studies with cytokines has decreased notably in recent chemo- or endocrine therapy.
334 A. Nicolini et al. / Cytokine & Growth Factor Reviews 17 (2006) 325–337

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for metastatic, hormone-sensitive breast cancer: An Eastern Coop- Medicine and Clinical Oncology for academic Institutes besides he is
erative Oncology Group study. J Clin Oncol 2000;18:262–6. scientific advisor of Biomedicine and Pharmacotherapy and regularly has
[112] Lindsey KR, Rosenberg SA, Sherry RM. Impact of the number of served as reviewer for many scientific Journals.
treatment courses on the clinical response of patients who receive
high-dose bolus interleukin-2. J Clin Oncol 2000;18:1954–9. Angelo Carpi is Clinical Researcher at the Uni-
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strategies with chemotherapy and biological response modifiers. Eur Professor of Internal Medicine, Pisa University,
J Cancer Clin Oncol 1989;S31–6. 1984 to today. Vice-Director Postgraduate
[114] Motzer RJ, Mazumdar M, Bacik J, Russo P, Berg WJ, Metz EM. School of Endocrinology, 1997 to 2003, Medical
Effect of cytokine therapy on survival for patients with advanced Faculty, University of Pisa. About 200 scientific
renal cell carcinoma. J Clin Oncol 2000;18:1928–35. publications in peer-reviewed journals. Principal
[115] Greenberg PA, Hortobagyi GN, Smith TL, Ziegler LD, Frye DK, fields: thyroid and breast tumours, male inferti-
Buzdar AU. Long-term follow-up of patients with complete remis- lity, vascular endothelium. Editor of Biomedicine
sion following combination chemotherapy for metastatic breast & Pharmacotherapy. Reviewer for International
cancer. J Clin Oncol 1996;14:2197–205. Journals with impact factor mainly in the fields of internal medicine and
[116] Atzpodien J, Korfer A, Franks CR, Poliwoda H, Kirchner H. Home endocrinology. Identified as international ‘recognised authority’ by the
therapy with recombinant interleukin-2 and interferon-alpha 2b in UCLA School of Los Angeles and the Faculty of Medicine of the Haifa
advanced human malignancies. Lancet 1990;335:1509–12. University. Recently involved in research on biomaterials, director of a unit
Andrea Nicolini is Clinical Researcher at the in a project of the CNR (National Research Council) on biomaterials.
Department of Internal Medicine at Pisa Univer-
sity. He was winner of fellowships at the Eur- Dr. Giuseppe Rossi obtained his B.Sc. degree
opean School of Oncology and of a grant by from the University of Pisa in 1978 and his PhD
Cancer Prevention and Detection Journal. His (Medical Statistics) in 1983 from the University
research over the past 15 years was mainly of Pavia. He currently is a Senior Scientist at the
devoted to the field of breast cancer, in particular National Research Council (Unit of Epidemiol-
to the use of serum tumour markers in the ogy and Biostatistics – Institute of Clinical Phy-
‘‘early’’ detection and treatment of metastatic siology) and Professor of Statistics in Medicine at
disease and to the function of cell mediated the University of Pisa. His research is focused on
immunity. Another area of research was the role of needle aspiration clinical and environmental epidemiology. Dr.
techniques and the use of tissue tumour markers (galectin 3) in the Rossi has published about 100 original papers
preoperative diagnosis of thyroid cancer. Dr. Nicolini has published about (including chapters of books), most of them in peer reviewed Journals. He
200 original papers (including chapters of books), most of them in peer served as secretary and treasurer of the Italian Region of the International
reviewed Journals and many review articles. He is teacher of Internal Biometric Society.