Dendritic cell immunotherapy for breast cancer

Review

10.1517/14712598.6.6.591 © 2006 Informa UK Ltd ISSN 1471-2598 591

Cell- & Tissue-based Therapy

Dendritic cell immunotherapy for breast cancerAlberto Pinzon-Charry, Chris Schmidt & José Alejandro López†

†Queensland Institute of Medical Research, Dendritic Cell and Cancer Laboratory, Royal Brisbane Hospital Post Office, Brisbane 4029, Australia

Novel adjuvant therapies are urgently needed to complement the existingtreatment options for breast cancer. The advent of the use of dendritic cells(DCs) for cancer immunotherapy provides a unique opportunity to overcomethe relative non-immunogenic property of breast tumours and address theunderlying immunodeficiency. To date, the success of this approach has beenlimited, possibly due to the targeting of specific tumour antigens that rapidlymutate and, thus, become undetectable to the immune system. A moreefficient approach would include preparations encompassing multipleantigens, such as those provided by loading of whole tumour cells or tumourRNA. It is proposed that targeting mammary stem cells responsible forresistance to chemo/immunotherapy, through the expression of a broad arrayof wild-type and mutated tumour antigens in the context of DCs, will becomea mainstay for immunotherapy of breast cancer.

Keywords: adjuvant therapy, breast cancer, dendritic cells, immunotherapy, mRNA, stem cells

Expert Opin. Biol. Ther. (2006) 6(6):591-604

1. Introduction

Over the centuries, treatment and management of breast cancer has remained closelyintertwined with our understanding of the disease. Tumours of the breast were firstdescribed by the Egyptians 300 years BC. Greek and Roman physicians wrote aboutthe disease, and the record continued through the middle ages into modern times.Notably, during the second century, Galen advanced the theory that disease wascaused by humoral imbalances, thereby influencing treatment for at least 1000 years.During this period, breast cancer remained conceptually a systemic disease caused byaberrations of body fluids, components of the blood, excess acid or alkali. Treatmentwas discouraged and tumours were recognised and left undisturbed. Subsequentprogress in anatomical knowledge and the discovery of the lymphatic system in the16th and 17th centuries brought about the concept that breast cancer was a diseasearising locally, thus raising hope for surgical approaches [1].

The first wide surgical resection of breast tumour together with excision ofaxillary lymph nodes were put forward in the early 19th century, and radical en blocmastectomies were later introduced as the first-line of treatment for patients withbreast cancer [1]. These approaches based on extensive surgical resections remainedprevalent and lasted as the standard treatment until the first half of the 20th century.Despite the widespread application of these techniques, death rates from breastcancer remained unchanged. Therefore, interest turned from maximising surgicalresection to the addition of adjuvant therapies, such as radiotherapy, chemotherapyand/or hormone therapy, for the control of local and systemic disease [2,3].

Significant progress has been made in this field. New cytotoxic agents, consolida-tion chemotherapy after primary postoperative chemotherapy, and preoperativechemotherapy have all shown enhanced therapeutic benefit for breast cancerpatients [4]. Nonetheless, tumour relapse, the emergence of toxicities, and early

1. Introduction

2. Immune response to

breast cancer

3. Target antigens for

breast cancer

4. Dendritic cells in

cancer immunotherapy

5. The use of dendritic cells in

breast cancer

6. In vivo interventions with

dendritic cells in breast cancer

7. Foreseeable therapeutic

combinations

8. Conclusion

9. Expert opinion

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Dendritic cell immunotherapy for breast cancer

592 Expert Opin. Biol. Ther. (2006) 6(6)

death due to the emergence of resistant disease continue topose great challenges for the clinical management of thisdisease. Hence, there is an urgent need for novel interventionstrategies capable of:

• synergising with standard adjuvant therapies• maintaining activity against refractory tumours• avoiding further immune suppression

Immunotherapeutic strategies that successfully activate theimmune system against tumour antigens are a prime alternativeas they would probably be non-cross-resistant, specifically boostantitumour immunity and have non-overlapping toxicities.

Over the last decade, the convergence of several lines ofresearch has bolstered interest in the use of dendritic cells(DCs) as the prime platform for immune therapy of breastcancer. One of the critical developments in the field has beena better understanding of the central role of DCs for the initi-ation of antitumour immunity. Ample evidence now indicatesthe existence of an immune response against breast cancerinvolving DC recruitment and activation within tumourtissue [5-7]. Our increasing understanding of the biology ofDC–tumour interactions in patients with breast cancer [8-10]

has also facilitated the development of more effectivetechniques for antigen delivery and the generation ofsufficient numbers of functional DCs for clinicalimplementation [11]. Finally, the molecular characterisation oftumour-associated antigens has permitted the identification ofa number of potential targets for immunotherapy [12]. Thus,DC-based vaccines are emerging as a crucial strategy toenhance therapeutic alternatives for patients with this disease.The efficacy of such a vaccine depends on its capacity toovercome tolerance by rendering weak immune responses to‘self tumour antigens’ strongly immunogenic, therebygenerating vigorous antitumour immunity.

2. Immune response to breast cancer

Breast cancer is an unusual disease amongst cancers. Unlikemost other malignancies, in which survival curves plateauwithin 2 – 5 years, survival curves in breast cancer require7 – 10 years to plateau depending on the stage of diseaseand the form of treatment [13]. These data not only indicatevariable rates of recurrence irrespective of type of treatment,but also suggest that host factors independent of the grade ofmalignancy and the extent of locoregional invasion play animportant role in determining the clinical outcome of thedisease. Similarly, the finding that from the 40% of patientswith involved lymph nodes not removed by surgery only15% recur [14] suggests that some of the malignant lesionsfail to progress, or regress, as a result of protectivemechanisms in the body. If it is accepted that persistenttumour cells can exist, but do not always cause recurrence,one must ask whether tumour immune recognition by thehost provides one of the most important components to thebiology of this disease.

2.1 Immune recognition of the tumourDue to their crucial role as effectors in the elimination oftumour cells, much research has been focused on assessinglymphocyte reactivity in patients with cancer. Indeed, theearly observation that primary tumour lymphocytic infiltra-tion correlated with a more favourable prognosis in patientswith medullary breast carcinoma [13,15] indicated a prominentrole for lymphocytes in the clinical outcome of this disease. Acomprehensive study of > 1900 breast tumours confirmedthat lymphocytic infiltration was a predictor of prognosis andsurvival, independent of tumour size and nodal status, inwomen who were younger than 40 years of age [16]. Giventhat tumours with more intense lymphocytic infiltrationassociate with a more favourable prognosis, one could suggestthat the host’s immune system within the tumourenvironment has the potential to recognise and impede thegrowth of neoplastic lesions. In accordance with this,tumour-infiltrating lymphocytes isolated from breast cancerpatients have been demonstrated to exhibit strong antitumourcytotoxicity after in vitro culture [17].

In addition to local immune reaction, systemic recognitionof breast tumours has also been reported. A proportion ofpatients with breast cancer have detectable T cell and/orantibody responses to the HER2/neu protein in peripheralblood [18]. Similarly, MUC1-specific cytotoxic T lymphocytes(CTLs) have been isolated from patients with adenocarcinomasof the breast that overexpress this mucin [19], and peripheralblood- and bone marrow-resident T lymphocytes isolated frombreast cancer patients have been shown to exhibit robusttumour-specific cytolysis following in vitro restimulation [20-22].Although these data suggest reactivity against breast tumours, aclear correlation between systemic immune responses andclinical outcome has been more difficult to ascertain. Forexample, MUC1-specific humoral responses correlate withprolonged disease-free survival [23]; however, HER2/neuhumoral and cellular immunity correlates with more aggressivedisease and, thus, poorer outcome [24].

Although the evidence remains ambiguous, if unrestrictedtumour growth occurs in the presence of effector lymphocyteresponses capable of tumour killing, then the immuneresponse elicited in patients with breast cancer isnon-functional or the tumour has the ability to evade thehost’s protective efforts. Indeed, a number of studies havedemonstrated that tumour-infiltrating lymphocytes in45 – 60% of breast cancers produce immunoregulatorycytokines, such as IL-4, IL-10 and/or transforming growthfactor (TGF)-β [25,26], thus negatively influencing the genera-tion of protective antitumour responses. Regulatory T cell(Treg)-mediated immune suppression has also beensuggested [27]. The frequency of Tregs in peripheral blood ofpatients with breast cancer has been found to be significantlyhigher than in healthy volunteers, and such accumulationappears to have an impact on the efficiency of the immuneresponse [27,28]. The interpretation of such data, however,should be cautioned by the need of a better definition of Tregs

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Pinzon-Charry, Schmidt & López

Expert Opin. Biol. Ther. (2006) 6(6) 593

using Foxp3 expression. For example, a recent report hasshown increased proportion of Foxp3-positive cells in breastcancer tissue, although not correlating with disease severity [29].Other mechanisms of evasion, such as induction of lymphocyteapoptosis, also play a role in tumour escape from immunesurveillance. In fact, overexpression of tumour cell-bound orsoluble Fas ligand has been proposed to contribute to theselective induction of T lymphocyte apoptosis in breast cancerpatients [30,31], confirming increased lymphocyte turnover rateand, thus, impaired immune competence.

2.2 Dendritic cell-mediated immunosuppressionGiven their central role in the generation of humoral andcellular responses, including antitumour immunity, there hasalso been enormous interest in the biology of tumour–DCinteractions in patients with breast cancer; a detailed review ofthe multiple tumour products affecting DC performance hasbeen published elsewhere [32]. Initial studies evaluated thefrequency of circulating as well as infiltrating DCs in breasttumours. Although blood DC numbers were reported to bealtered in patients with advanced disease [33], tumour-infiltratingDCs were readily detected in > 40% of patients with early andadvanced breast cancer [5]. Such studies indicated a relationshipbetween DC infiltrate and tumour size, lymph node involve-ment, oestrogen/progesterone receptor status and age. However,the presence of DCs, even at the highest density, was notcorrelated with metastasis-free survival or overall survival.

Subsequent studies indicated a direct involvement of breasttumours in DC suppression. Indeed, it was demonstrated thattumour-infiltrating DCs were immature, whereas DCs in thesurrounding stromal tissue adopted a more maturephenotype [7]. Such compartmentalisation suggested thatbreast cancers could actively recruit immature DCs to thetumour site, but subsequently impede their differentiationinto functional antigen-presenting cells (APCs). Importantly,clusters of mature DCs in the peritumoural area wereassociated with T lymphocyte infiltration [7] and lowerexpression of intratumoural inhibitory cytokines, such asvascular endothelial growth factor or TGF-β [6], and this, inturn, correlated inversely with lymph node status andimpacted favourably on survival.

Despite this evidence, numerous studies have demonstratedsevere phenotypic and functional impairment of DCs inpatients with breast cancer [8,34]. Circulating DCs isolated frompatients with breast cancer exhibit an impaired capacity tostimulate T lymphocyte proliferation and cytokinesecretion [8], express low levels of costimulatory molecules [8,34],and display altered maturation profile and reduced secretion ofIL-12 in response to maturation stimuli [9]. Moreover, patientswith metastatic breast cancer show a large number of immatureAPCs with poor immunological function in the blood DCcompartment [35]. These immature cells displace bona fide DCpopulations and appear to have an impact on the nature of theimmune response observed in these patients. Of interest, thefunctional capacity of these immature populations can be

restored by ligation of the CD40 molecules on theirsurface [36]. These findings are particularly relevant in thecontext of maturation signals for DC immunotherapy(discussed below). There is also evidence of increased apoptosisof circulating DCs in patients with early-stage breastcancer [10]. DC apoptosis was shown to correlate withtumour-derived products that presumably gain access into thecirculation. Importantly, continual efforts to replace the pool ofDCs would impose chronic stress on the immune system,resulting in failure to replenish DC populations in blood andtissue, thus favouring tumour evasion. Interestingly, in vitroevaluation showed that this process of apoptosis could also bereverted/prevented by CD40 ligation [10].

These data indicate that many influences, includingtumour-derived products, are capable of affecting immunecells (T and B lymphocytes and DCs), thus exerting systemiceffects on immune function and affecting clinical outcome.Although a better understanding of the biology of theseDC–tumour interactions in patients with breast cancer willfacilitate the development of more effective therapies, theimplementation of a successful DC immunotherapeuticapproach for breast cancer also hinges on the identification ofthe appropriate target antigens.

3. Target antigens for breast cancer

The molecular characterisation of several tumour-associatedantigens (TAAs) to the level of the specific MHC-restrictedepitopes in other malignancies, such as melanoma and renalcell carcinoma, has added momentum to the evaluation ofDC-based immune approaches for breast cancer. TAAsrelevant to epithelial tumours have generally been postulatedon the basis of either differential expression studies (particu-larly for antigens already identified in other tumour systems)or serological identification of antigens by recombinantexpression cloning (SEREX) [12]. The final designation asTAA then requires demonstration that some patients possessintratumoural or peripheral T cells capable of recognisingendogenously expressed TAAs in an MHC-restricted fashion.Breast TAAs defined so far are involved in tissue-differentia-tion (derived from proteins involved in embryonic differentia-tion), overexpressed in breast cancer (functional molecules ofadult tissue found in higher than normal levels) oroverexpressed in multiple tumour types (shared), includingproteins involved in the process of malignant transformation.Breast cancers from different patients express varying levels ofTAAs (Table 1), and this heterogeneity often extends toindividual tumours. Thus, no single-antigen vaccine is likelyto be applicable to all patients, and even cancers expressingthe relevant TAAs may harbour resistant cells that allow thetumour to evade any elicited immune response.

3.1 MucinOne of the most studied target antigens in breast cancer isMUC1, a heavily glycosylated cell surface mucin normally

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polarised to the apical surface of epithelial cells. It is anattractive tumour antigen as in neoplastic cells thecarbohydrate side chains are shorter and the amino acidbackbone is more exposed [37]. Moreover, MUC1 is present ingreatly increased amounts and has high cell surface expressionin tumour cells. MUC1 expression is increased up to 10-foldin breast tissue and is present in 90% of breast tumours [38].Importantly, MUC1 recognition by classical MHC-restrictedand -unrestricted CTLs, as well as by anti-MUC1 immunecomplexes, has been documented [19,39]. Increased IgG andIgM antibody responses to mucin have been correlated withprolonged disease-free interval and overall survival in womenwith early-stage disease [23], implicating immune responses toMUC1 in protective immunity. MUC1-specific CTLscapable of eliminating xenotransplanted autologous breast

tumours in mice have been identified both in bone marrowand peripheral blood of breast cancer patients [40]. TheseCTLs were found to be activated memory cells, indicating theinduction of a long-lasting effector mechanism available toeliminate the tumour [41]. These findings substantiate theenormous interest posed in MUC1-based vaccines.

3.2 HER-2/neuOne of the first TAAs described for breast cancer was theencoded protein of the HER-2/neu oncogene a transmembraneglycoprotein from the epidermal growth factor receptor family,implicated in the pathogenesis. Extensive research on thismolecule has led to a monoclonal antibody widely usedclinically as an adjuvant therapy [42]. HER-2/neu is expressedat low levels on normal tissue, but overexpressed in 20 – 30%of breast cancers [43], and its expression correlated with theaggressiveness of the disease and poor prognosis. Importantly,cellular and humoral immune responses to this protein havebeen detected in patients with breast cancer [18,24], andpre-existing immunity has been shown to be amenable toenhancement via immunotherapeutic approaches [44].

3.3 TelomeraseThere are other target antigens postulated for use in breastcancer that are shared with other malignancies. One of them,human telomerase reverse transcriptase (hTERT) is a cellularreverse transcriptase responsible for maintenance of telomerelength and, thus, chromosomal stability [45]. Unlike normalmammary tissue where expression is absent, hTERT isexpressed in > 90% of breast tumours [45]. Overexpressionmakes this TAA an appealing molecular target, particularly asexpression of hTERT in breast cancer tissue correlates withpoor prognosis [46,47]. Interestingly, 75% of patients withbreast cancer have been reported to carry hTERT-specificCTLs [47], indicating the potential of this enzyme as a suitabletarget for immunotherapy.

3.4 p53p53 is a tumour suppressor gene involved in DNA transcrip-tion and repair. Mutations in this gene have been found inmany common malignancies, and it is overexpressed in > 50%of human cancers. Mutations have been detected in highproportion breast cancer lines and primary tumours [48], andthese correlate with poorer outcomes. Moreover, theoverexpression of p53 in multiple cancers has made it anattractive candidate for immunotherapy, and specific CTLsagainst the wild-type sequence have been established frompatients and healthy donors using DCs [49].

3.5 SurvivinAnother shared TAA of interest is survivin, a central memberof the inhibitor of apoptosis protein family. As with telomeraseexpression, survivin expression in normal breast tissue is low orabsent, whereas it is increased in breast tumours [50].Importantly, in 20 – 60% of patients with breast cancer, CTL

Table 1. Breast cancer antigens.

Target antigen Expression (%)

Immune response Refs.

Tissue-differentiation Carcinoembryonic antigen (CEA)

50 humoral and cellular [107]

NY-BR-1 80 humoral [108]

NY-ESO-1 24 humoral and cellular [109]

MAGE3 14 humoral and cellular [109]

SCP-1 31 humoral [109]

SSX-1 12 humoral [109]

SSX-2 8 humoral [109]

SSX-4 14 humoral [109,110]

CT-7 30 humoral [110]

Overexpressed in breast cancer

HER-2/neu 40 humoral and cellular [18]

MUC1 80 humoral and cellular [19,39]



D52 60 humoral [111]

Mammaglobin 23 humoral and cellular [112]

BA46 (Lactadherin)

NA cellular [52,113]

Folate binding protein

20 – 50 cellular [53]

Shared (non-breast-specific)Telomerase 70 humoral and cellular [45]

Survivin 20 – 60 humoral and cellular [50]

p53 17 cellular [48]

Potential target antigens for immunotherapy of breast cancer, described on the basis of their pattern and frequency of expression, as well as type of immune response generated.BA46 is abundantly found in milk and detected in breast cancer cell lines.NA: Not analysed.

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responses against survivin have been detected both inperipheral blood and tumour tissue, whereas responses inhealthy individuals have not been documented [51].

3.6 Other antigensMore recently, using DCs as a tool for the investigation ofnovel potential antigens, BA46, also known as lactadherin(milk fat globular membrane), a protein abundant in milkand identified in breast cancer cell lines, has beendemonstrated to be immunogenic [52]. Similarly, DCs havebeen instrumental in the evaluation of the folate bindingprotein as potential novel breast cancer antigen [53].

Whereas in other malignancies tumour tissue has beenused as a source of a comprehensive collection of antigens,this option is not usually feasible in breast cancer. With theadvent of early diagnosis and treatment, most patients withoperable breast cancer exhibit small primary tumours (often< 2 cm), and patients with metastatic disease do not havereadily accessible lesions (brain, bone, liver) that could besurgically removed to provide for antigen preparation. Inaddition, the importance of studying primary tumour anddraining lymph nodes for clinical staging linked totherapeutic decisions further limits tissue access. Therefore,techniques such as T cell epitope cloning, SEREX andgenomic approaches [12] are proving essential for thisendeavour, and the identification of novel target antigens forbreast cancer is already in progress (Table 1).

4. Dendritic cells in cancer immunotherapy

As mentioned above, since the ‘rediscovery’ of DCs a coupleof decades ago, their potential as initiators of immuneresponses has been extensively studied [54]. Their ability toelicit responses against novel and self antigens made themobvious candidates to be tested for cancer immunotherapy;hence, the growing interest for the use of DCs in clinicaltrials [55]. However, to date, the clinical outcome of thevarious trials has not met these high expectations, althoughsome encouraging results have been seen. Here, the issueslikely to improve the clinical results are discussed.

4.1 What do we know about effective antitumour immunity?Currently licensed active human vaccines, even those usinglive attenuated viruses, appear to rely largely on antibodies forlong-term protection [56]. Therefore, outside of mousemodels, there exists little precedence for adjuvants effective ateliciting specific, long-lived CD8+ T cell responses in vivo,even against foreign antigens. The debate at present on the useof DCs for cancer vaccination needs to be seen in this light:most clinical trials are based on vaccines formulated to mimic‘successful’ murine models rather than building on effectivepre-existing vaccines. However, as a further caveat, the goal ofcuring aggressive transplantable tumours in mouse models hasalso rarely been achieved [57,58], even in the presence of strong

antitumour cellular immune responses. Nonetheless, humanclinical trials have yielded a small but fairly consistentpercentage of objective clinical responses to immunotherapy,particularly in the case of DC-based therapy of melanoma [59].

4.2 Barriers to effective cancer responsesWhat does appear clear from some of the in vivo preclinicalstudies is that disrupting the normal or tumour-inducedimmunoregulatory environment can greatly enhance tumourclearance [60,61], and systemic agents targeted to regulatorymechanisms have been applied clinically to increaseantitumour T cell activity [62,63]. Immune suppression at thelevel of host DCs, such as that detailed earlier, might alsolimit the potential of cancer vaccines in which antigens areinjected in the form of proteins or nucleic acids mixed withadjuvants. This can potentially be overcome by culturing DCprecursors ex vivo, the approach adopted by cancer trialsemploying CD34+-derived or monocyte-derived DCs(MoDCs). Immature MoDCs from patients with breastcancer have been noted to be functionally and phenotypicallydifferent to those from healthy women [64]. As noted, furtherconditioning with maturation or activation agents, such asCD40L, might in addition restore function of dysfunctionalAPCs in cancer patients [36].

4.3 Antigen sourcesWith notable exceptions [65], most murine vaccine studiesemphasise the need for both CD8+ effector and Th1-polarisedCD4+ helper responses for effective antitumourimmunity [66]. The target for the helper T cells in primaryimmune responses does not need to involve the target(tumour) antigen [67]. Consequently, the source of helpemployed in some DC trials is an irrelevant, foreign antigen,such as keyhole limpet haemocyanin (KLH). This is particu-larly the case in the many trials employing synthetic peptidesrepresenting HLA class I-restricted TAA epitopes. Conven-iently, this also allows a measurement of the effectiveness ofthe DCs at inducing T cell responses against a foreign protein.As tolerance of existing tumour cells may be the normalstate [68], most active vaccination protocols also invoke repeti-tive injections. This introduces the potential for repeatedvaccination against any contaminating foreign protein (e.g.,KLH) to induce very strong CD8+ and CD4+ T cell responsesthat might dominate the lower affinity (due to deletionaltolerance) antiself responses desired to target the cancer. Theincreased number of lymphocytes elicited may potentiallylead to a homeostatic downregulation of antigen presentation,for example, by killing the injected DCs [69], or the inductionof Tregs suppressing the function of the DCs. Whether this isa real problem in DC vaccination remains to be seen.

Complete and persistent clinical responses to DC vaccina-tion have been observed with both multiple antigen syntheticpeptide-pulsed DCs [70] and with vaccines using lysates orwhole tumour cells [71,72]. It is now clear that tumourregression following immunisation with peptide-based

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vaccines may be the result of epitope spreading, that is, aninitial antipeptide response facilitates the expansion ofresponses against other, non-targeted TAAs [73,74]. Severalstudies have also shown that targeting individual TAAs canlead to tumour escape by gene deletion ordownregulation [75,76]; thus, vaccines employing multipleTAAs are more likely to effect tumour eradication. Bothconserved and mutated proteins can serve as targets ofantitumour immune responses [77,78], and their relativeimportance in immunotherapy remains unknown [75,79].Evidently, the success of ‘off-the-shelf ’ vaccines for cancerdepends on the ability of responses against non-mutatedTAAs (Table 1) to induce complete clinical regression, so thisis a critical issue. Combining these observations, there is astrong case for using multiple TAAs from patients’ owntumour cells, whether intact, as lysates, or in the form ofRNA, as the source of tumour antigen for the DCs. Theseheterogeneous mixtures potentially contain or encode bothclass I- and class II-restricted TAA epitopes, as well as mutatedtumour-specific epitopes. The effectiveness of repeatedinjections of such vaccines may be still be limited by theantitumour CTLs or Tregs that they elicit, and the challengefor future DC/chemotherapy regimens is to reduce theundesired effects of such responses.

There is increasing evidence that cancers harbour anintermittently cycling progenitor stem cell populationresponsible for renewing the more differentiated (andnon-tumourigenic) cancer cells comprising the bulk of thetumour. As with standard chemo- and radiotherapies, thereremains the possibility that cancer stem cells are differentiallysensitive to immune attack, for example, via altered levels ofantigen presentation. This could arise due to a differentprofile of expression of differentiation or cancer testisantigens, potentially permitting escape from the dominantantitumour response elicited by the bulk of tumour cells.Alternatively, the progenitor population may differ in itsantigen-presenting capacity. It has been observed that rarecomplete clinical responses of solid tumours to immune-basedtherapies are generally long-lasting (> 5 years), in contrast tothe transient responses to chemotherapies [70,72]. If an antigen-ically distinct population of stem cells is indeed responsiblefor the resistance of solid tumours to standard therapies, thissuggests that they are potentially sensitive to the immuneresponse. The combination of debulking measures withimmune therapy to mop up residual stem cells may be theoptimal approach of using the most useful attributes of eachmodality. However, successful implementation would dependon the identification of the appropriate target antigens. Asnoted below, most T cell antigens defined for breast cancerhave been postulated on the basis of serological or expressionstudies, rather than deduced de novo from existing antitumourT cell responses of patients. The latter is difficult as tumourcell lines are not readily established from patients’ tumours.The recent description of ‘mammospheres’ [80], believed torepresent the breast equivalent of ‘neurospheres’ [81] and

containing the self-renewing progenitors of normal differenti-ated breast cells, may provide a novel resource for antigendiscovery in breast cancer patients.

4.4 Unanswered questionsAlthough a theoretical consideration [82], the potential ofeliciting deleterious autoimmune responses to additionalepitopes has not been documented in any of the numeroustrials reported to date [55,59]. While DC therapies might noteasily become ‘off-the-shelf ’ options due to operationalconstraints, if proven to be successful, the added financialburden will have to be tolerated, as has been the case for bonemarrow transplantation.

Other general aspects of DC immunotherapy that requirefurther understanding include the migratory and maturationstatus of DCs following injection, which will certainly play arole in the final effectiveness of the therapy. However, to date,there is little information on the influence of these parametersin humans.

5. The use of dendritic cells in breast cancer

Whereas significant dysfunction in the DC compartment ofpatients with breast cancer is well documented, their in vitrocounterparts generated from blood/bone marrow precursorsand transformed in the presence of cytokines appear to befunctional. This strategy has been tested both for monocyte- [8]

and CD34+-derived DCs [83]. Once isolated from theinhibitory environment of the patient’s circulation (and/ortumour tissue), precursors yield competent mature DCscapable of inducing immune responses to a level similar to thatachieved with DCs from healthy matched controls. CulturedDCs have been also evaluated for their potential to inducetumour-specific CTLs. Gong et al. demonstrated that fusionsof DCs with breast cancer tumours stimulated autologousCTLs capable of lysing autologous tumour cells [84]. Thisresult indicated that despite the inhibitory effect of the tumourtissue in vivo, close contact of tumour cells with DCs in thepresence of exogenous cytokines granulocyte-macrophagecolony-stimulating factor (GM-CSF) and IL-4 was notdeleterious for DC function. Lately, complementary work inan allogeneic system showed that healthy donor DCs loadedwith a killed breast cancer cell induced both CD4 and CD8responses, and yielded CTLs able to lyse target cells [85]. In thisstudy, DC maturation was achieved with CD40L, reminiscentof the DC function rescuing capacity of CD40 ligationobserved in patients with breast cancer [10,36]. A recent reportcomparing phenotype and function of DCs generated underclinical-grade conditions from healthy controls and patientswith advanced breast cancer showed that both DCs inducedsimilar levels of antigen-specific responses; however, theirresponsiveness to maturation stimuli varied [11]. Overall, thereports in the literature agree that, under the appropriatein vitro conditions, DC precursors can be made functional andcapable of inducing efficient immune responses.

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6. In vivo interventions with dendritic cells in breast cancer

Fuelled by the encouraging results with other solidmalignancies, in particular melanoma, as discussed earlier,various clinical immunotherapeutic interventions have beenattempted for patients with advanced breast cancer. Thegeneration of DCs from precursors in patients with breastcancer as been reported to be safe and yielding sufficientnumbers for immunotherapy. The majority of the studiesdiscussed here have used plastic-adherent monocytes obtainedfrom apheresis differentiated in the presence of GM-CSF andIL-4. Immunisation doses varied between 50,000 and30 million DCs and, as in other malignancies, the vaccinationprocedures have been well tolerated and no signs ofautoimmune reactions have been described. Table 2summarises the interventions reported in the literaturegrouped by the type of antigen used.

6.1 No antigenThe first intervention reported in patients with advancedbreast cancer was the intratumoural injection of a large dose(30 million DCs) of immature MoDCs [86]. This small trial(3 patients), although inconclusive, showed some regressionof the injected tumours and the immunohistopathology

evidence that DCs were functional in the tissues. Theimmunological outcome of this study was the induction ofproliferative responses to heat-shock proteins bytumour-infiltrating lymphocytes.

6.2 One specific antigenThe scarcity of TAAs in breast cancer is reflected in thelimited number of studies performed to date using singleantigens, that is, HER-2/neu, telomerase (hTERT), p53 orMUC1. As immunogens, most of the trials have opted to usea single antigen delivered in the form of synthetic peptidesrepresenting HLA-A*0201-restricted epitopes identified inpreclinical evaluations. The peptides used reflect either thewild-type sequence of the protein or altered ligand peptides,known to have better MHC binding capacity [83]. In one ofthe trials, a mutant variant of p53 was also used [87]. The DCpreparations were mainly of monocyte origin and wereinjected subcutaneously, except in a trial with HER-2/neupeptides where DCs were produced from CD34 precursorsand injected intravenously [83]. Amongst the MoDC prepara-tions, all but one trial utilised immature MoDCs, a source ofDCs reported to repress already established immune responsesand believed to induce tolerance [88]. The only trial in whichMoDCs were matured before injection used TNF-α as thematuration agent; in this particular trial, patients were

Table 2. In vivo studies of DC-based therapy for metastatic breast cancer in humans.

Antigen Delivery form DC Route Patients treated/clinical response

Immunological responsespecificity/type

Refs.

No antigen iMoDC intratumoural 3/2 tumour regressions

HSP/TIL proliferation [86]

HER-2/neu 2 peptides MoDC s.c. 5/inconclusive 3 peptide, 1 tumour/CD8

[73]

HER-2/neu Peptide and ALP CD34 DC i.v. 10/6 PD, 1 SD, 2 PR 2 peptide/CD8 [83]

hTERT I540 + KLH Peptide iMoDC s.c 2/2 PD Peptide and tumour/CD8

[114]

p53 3 wild-type + 3 mutant peptides

iMo-DC + IL-2 s.c. 6/2 SD, 3 PD 3 peptide/CD8 [87]

MUC1 2 peptides MoDC s.c. 2/inconclusive 2 peptide, 1 tumour/CD8

[73]

MUC1 cDNA liposomal transfection

iMoDC s.c. 7/1 SD, 6 PD 3 peptide/CD8 [89]

MUC1 Mannan + recombinant DNA

iMoDC s.c. 2/1 SD, 1 PD 2 protein-specific/ CD8, CD4

[90]

Autologous tumour + KLH

Lysate iMoDC intralymphatic 1/local metastatic regression

CD45+ CD3 lymph infiltrate

[93]

Autologous tumour + KLH

Fusion iMoDC s.c. 10/1 SD, 2 PR, 7 PD Lysate/CD4 (3) and CD8 (1)

[94]

ALP: Altered ligand peptide; DC: Dendritic cell; HSP: Heat-shock protein; hTERT: Human telomerase reverse transcriptase; iMo-DC: Immature monocyte-derived DC; i.v.: Intravenous; KLH: Keyhole limpet haemocyanin; MR: Mixed response; PD: Progressive disease; PR: Partial response; s.c.: Subcutaneous; SD: Stable disease; TIL: Tumour-infiltrating lymphocyte.

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injected with either MUC1 or HER-2/neu peptidesdepending on the level of expression of the tumours [73]. Inboth groups of patients, antitumour and peptide-specificresponses were successfully elicited. The overall clinicalresponses have been modest or inconclusive. Short-lastingstabilisation of the disease and partial responses were the bestoutcomes. Interestingly, in all of the trials reported, CD8lymphocyte responses were detected. The specificity of theseresponses was mainly determined using the immunisingpeptides, but in 3 of the 6 trials reported, activity againstbreast tumour cells was also identified.

One of the trials utilising a single antigen (MUC1)increased the number the epitopes delivered by loadingMoDCs with cDNA via liposomal transfection. However, avery wide range (2 – 56%, with average 21%) of transfectionefficiency rates was reported. Immunological responses,measured as IFN-γ production by CD8 lymphocytesstimulated with synthetic peptides, were detected in 3 of7 patients and did not correlate with clinical outcome [89].More recently, a novel approach to deliver MUC1 DNA toDCs via mannan was used in a series of patients with variousadenocarcinomas. Protein-specific responses were detected inthe 2 patients with breast cancer, and reactivity was found inboth CD4 and CD8 lymphocytes in one patient. However,clinical responses were modest [90].

The use of genetically modified DCs for the therapy ofbreast cancer has been further investigated in preclinicalevaluations. MoDCs retrovirally transfected with HER-2/neuexpanded strong CD4 and CD8 lymphocytes recognisingHER2/neu-overexpressing tumours [91]. This approach hasalso been investigated in a murine model in which humanHER-2/neu transgenic mice were prevented from developingmammary tumours following vaccination with HER-2/neuadenovirus-transduced DCs [92].

6.3 Whole tumourOne of the first reports on the use of DCs for immunisationin advanced cancer utilised a lysate of autologous tumour inthe presence of KLH in a single patient. Intralymphatic(radiological guided) injection of repeated doses oflysate-loaded immature DCs yielded limited regression of themetastases local to the injection site. Interestingly, theyexhibited significantly higher CD3 CD45+ lymphocyteinfiltrates than distant metastasis [93].

Following the preclinical evaluation described above,Avigan et al. performed a Phase I clinical trail with fusions ofautologous tumour and DCs in patients with breast andrenal cancer [94]. Ten patients with advanced cancer wereevaluated. Polyethylene glycol-mediated fusions oftumour/DCs at ratios of 1:3 – 1:10 were > 46% viable, andthe efficiency of fusion was in the range of 29 – 70%. Onepatient had disease stabilisation coinciding with both CD4and CD8 recognition of tumour lysates. Although wholetumours potentially provide multiple target antigens, thelimited source of breast cancer tissue available for vaccine

preparation makes this a very challenging strategy. Antigenloading with RNA may provide the solution. Using wholetumour RNA-loaded DCs, encouraging clinical outcomeswere reported in patients with metastatic renal cellcarcinoma. CTLs specific for multiple antigens were elicited,capable of lysing autologous tumour cells. Although theclinical outcome was difficult to evaluate due to the use ofconcurrent chemotherapy, there appeared to be a lowermortality rate in patients receiving the treatment [95].

7. Foreseeable therapeutic combinations

The trials reported to date have evaluated DC therapy inpatients in whom successive treatments failed to provideclinical improvement. One trial examined the effect of DCimmunotherapy combined with systemic IL-2 [87], but nomethodical attempt has yet been made to combine existingsurgery/chemo/radiotherapy with DCs. The potentialbenefits of prior or concomitant chemotherapy could includethe elimination of suppressor factors produced by the tumour,elimination of potential Treg lymphocytes and their capacityto modulate of cytokine secretion, yielding a more conduciveenvironment for lymphocyte activation, which wouldfacilitate lymphocyte homeostasis. The increased availabilityof apoptotic tumour cells could provide strongmaturation/differentiation stimuli to DCs, and angiogenesismay also be favoured [96,97]. Administration of this combina-tion therapy needs to accommodate sufficient time for therecovery of the proliferative potential of lymphocytes. Indeed,the evaluation of coordinated radio-chemotherapy with othertypes of breast cancer immunotherapy (e.g., monoclonalantibodies) in clinical studies and animal models suggests thatit may improve efficiency [98,99].

Postvaccination chemotherapy has been examined. In anon-randomised comparison of glioblastoma patients treatedwith standard chemotherapy, or with DC vaccination withoutsubsequent chemotherapy, or vaccination followed bychemotherapy at the time of tumour progression,Wheeler et al. [100] noted a significant improvement insurvival in the latter group.

8. Conclusion

The promising success observed in DC trials in othermalignancies has been difficult to translate in the treatmentof breast cancer. However, the encouraging results derivedfrom the DC-based therapy of advanced melanoma suggestthat successful implementation of DC immunotherapy forbreast cancer is feasible providing the following issues areconsidered. First, the source of antigen. Given that bothwild-type and mutant antigens may play a key role in theimmune response eliminating the tumour, a comprehensivesource of antigen (e.g., whole tumour or tumour RNA)capable of inducing both CD4 and CD8 responses,containing multiple epitopes, restricted by multiple MHC

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molecules and representing multiple antigens should befavoured. Second, the choice of DC. Overcoming thedysfunction of circulating DCs could be obtained by in vitrodifferentiation of precursors yielding fully functional DCs.These DCs should be mature, so as to eliminate thepotential of the induction of tolerance and provide strongerand long-lasting immune responses. It has beendemonstrated that the use of immature DCs may lead totolerance and loss of established CTL responses [88].Appropriate maturation reagents for DCs from patients withadvanced cancer should be explored, and they may welldiffer from those optimal for DCs derived from patientswith minimal residual disease or healthy individuals. Finally,the relative benefits of a fully autologous system thatprecludes the potential for induction of distractingnon-tumour-specific responses should also be investigated.

Whether the reduction of tumour load is achieved bysurgery or by chemo/radiotherapy, an immune response islikely to be more effective against minimal residual disease.Although DC immunotherapy has had encouraging results ina proportion of patients with advanced malignancies, fewstudies have been performed in the adjuvant setting. Theimplementation of DC adjuvant therapy for breast cancer islikely to be in combination with other modalities, and willrequire randomised studies to determine whether immuno-therapy provides an additional benefit. Thorough evaluationof its efficacy, in turn, will require development of standard-ised protocols. Given the myriad of variables of demonstratedrelevance, for example, type of antigen and antigen loading, aswell as choice of DC and delivery method, areas of consensusneed to be defined. This will assure that this type of therapymoves ahead from defined patient study series to successfulimplementation as a therapeutic alternative for patients withbreast cancer.

9. Expert opinion

The data gathered in this review highlight the potential ofDC-based immunotherapy for breast cancer and the ways inwhich it can be improved. Dysfunctional DCs derived frompatients may be improved by exogenous maturation agents.The main obstacle to overcome remains the appropriatesource of autologous antigen, that is, representing multipleantigens presented in the context of multiple MHC I and IImolecules. Where whole tumour preparations are not analternative, transfection of DCs with amplified

tumour-derived nucleic acids is a viable option. Asdemonstrated in other malignancies, mRNA-transfected DCsare capable of inducing CD4 and CD8 antitumour responses,while avoiding the delicate regulatory and safety issues linkedto gene therapies [101,102]. Improved clinical-grade techniquesfor the introduction of RNA onto DCs will probably translateinto better outcome. In addition, the efficiency of DC-basedimmunotherapy is likely to be optimal in the minimalresidual disease setting and enhanced by the coordinated useof radio/chemotherapy adjuvant regimens.

A very interesting new development in our understandingof malignancies is the identification of cancer stem cells. It isbelieved that a small group of these cells that arise at thebeginning of the tumour differentiation process carrying themutated and altered phenotype of the tissue of origin wouldbe responsible for the features of uncontrolled growth, drugresistance and unlimited self replication [103]. These cells canbe isolated and cultured following a technique developed forthe study of adult neural stem cells in the form ofneurospheres [81]. It has now been possible to produce‘mammospheres’ from healthy tissue [80] and, recently,mammary epithelial stem cells have been purified andcharacterised [104]. While the definitive description of breastcancer stem cells awaits confirmation, tumourigenic breastcancer cells with stem/progenitor cell properties have beendescribed [105]. The implications of the existence of stem cellsat the origin of breast cancer on therapeutic and prophylacticstrategies has been extensively discussed [106].

We propose that an ideal source of antigen for DCimmunotherapy will be the breast cancer stem cells, providingall antigens likely to be expressed in the tumour. The ability toextract RNA from these cells and to efficiently load it ontoDCs will provide a very powerful tool for breast cancerimmunotherapy. By targeting the self-replicating cell at theorigin of drug resistance, a combined therapeutic approachincluding surgery, chemo/radiotherapy and immunotherapyis more likely to succeed.

Acknowledgements

All three authors contributed equally to this work. AP-C hasbeen supported by the University of Queensland Interna-tional Postgraduate Research and the Paul Mackay BoltonCancer Research Scholarships. CS is supported byNH & MRC, Australia. JAL is supported by the NationalBreast Cancer Foundation, Australia.

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• This is the first description of the technology leading to the culture (isolation) of tissue-specific (brain) stem cells. The methodology is now applied to culture breast tissue.

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•• Although the clinical outcome of this approach is limited, it demonstrates the feasibility of using DCs loaded with allogeneic whole tumour cell preparations for breast cancer immunotherapy capable of eliciting tumour-reactive CTLs.

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• This was the first report of DC interventions in patients with breast cancer resulting in some measurable effect.

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87. SVANE IM, PEDERSEN AE, JOHNSEN HE et al.: Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer: report from a Phase I study. Cancer Immunol. Immunother. (2004) 53(7):633-641.

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•• This key report highlights the danger of utilising immature DCs for immunotherapy as they inhibit antigen-specific CTL activity. Unfortunately, most clinical trials for breast cancer have used immature DCs.

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•• This group has pioneered the use of DC–tumour fusions and have shown that

they can generate immunological and clinical responses.

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•• This is a detailed discussion on the mutually adjuvant effects of chemotherapy and immunotherapy for the treatment of breast cancer.

98. YU B, KUSMARTSEV S, CHENG F et al.: Effective combination of chemotherapy and dendritic cell administration for the treatment of advanced-stage experimental breast cancer. Clin. Cancer Res. (2003) 9(1):285-294.

• This report highlights the potential of combination therapies, including DC therapy and chemotherapy, in a mouse model.

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100. WHEELER CJ, DAS A, LIU G, YU JS, BLACK KL: Clinical responsiveness of glioblastoma multiforme to chemotherapy after vaccination. Clin. Cancer Res. (2004) 10(16):5316-5326.

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• A recent review of the application of RNA technology for DC loading in cancer immunotherapy.

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•• A confirmation of the stem cell properties of cells isolated from mammary epithelia tissue in mice.

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AffiliationAlberto Pinzon-Charry1, Chris Schmidt2 & José Alejandro López†3

†Author for correspondence1Queensland Institute of Medical Research, Dendritic Cell and Cancer Laboratory/Molecular Immunology Laboratory, Brisbane 4006, Australia2Queensland Institute of Medical Research, Cancer Immunotherapy Laboratory, Brisbane 4006, Australia3Queensland Institute of Medical Research, Dendritic Cell and Cancer Laboratory, Royal Brisbane Hospital Post Office, Brisbane 4029, AustraliaTel: +61 7 3845 3794; Fax: +61 7 3845 3510;E-mail: [email protected]

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Dendritic cell immunotherapy for breast cancer