Download TAA loaded DCs did not consistently induce clinically relevant

Is Dendritic Cell Based Immunotherapy an Effective Treatment for
Colorectal Cancer?
Abstract
Colorectal cancer is a major malignancy with a sharply rising incidence rate. Recent advances
in surgery and chemotherapy have yielded modest treatment benefits, as approximately 50% of patients
develop recurrent or metastatic disease. Treatments based on using the patient’s own immune system to
target malignancy have gained attention, as tumour-specific immune responses can be mounted against
tumour-associated antigens (Ags). Dendritic cells (DCs), the most potent Ag presenting immune cells,
are an attractive mode of immunization, as they also produce co-stimulatory signals required to drive
the differentiation of quiescent T cells into Ag specific effector cells. As DCs derived from cancer
patients are minimally activated and deficient in co-stimulatory molecule expression, defined protocols
have been established to isolate autologous DCs from peripheral blood, for culture and exposure to
tumour associated Ags before re-injection into patients. In mouse tumour models, Ag loaded DCs were
able to initiate tumour-specific cytotoxic T cell responses, against both primary and metastasized
tumours. These encouraging findings have generated the hypothesis that tumour Ag loaded DCvaccination can be used to treat colorectal cancer. To date, studies have only been possible with small
numbers of patients with advanced colorectal cancer, who have increased tumour burden and are
immunosuppressed from chemotherapy. As the heterogeneity between these pilot studies precludes
systematic analysis of the data, the aim of this review is to critically analyze clinical data to
demonstrate that DC-based immunotherapy is a feasible treatment with potential therapeutic benefits
for colorectal cancer, in light of the described advantages and limitations of current vaccination
[1]
protocols. Determining whether DC-based immunotherapy has therapeutic potential will have
important implications for the millions of individuals suffering from colorectal cancer.
[2]
Introduction
Colorectal cancer is the second leading fatal malignancy in the Western world, and represents
approximately 10% of all incident cancer worldwide.1;2 Common treatments include surgical resection
of primary tumours and 5-fluorouracil based adjuvant chemotherapy (with or without radiation).2
However, approximately 50% of patients still develop recurrent or metastatic disease, and most die as a
consequence.1 As these treatments are limited by their toxicity and lack of tumour specificity,
interventions based on using the patient’s own immune system to target tumours have recently gained
attention. A rationale for implementing immunotherapy was also provided by the positive correlation
between lymphocytic infiltration at the tumour site and positive clinical outcomes for colorectal cancer
patients.2-4 Both humoural and cellular anti-tumour immune responses have previously been reported,2
with a key role for CD8+ cytotoxic T lymphocytes (CTLs) in recognition and lysis of tumour cells that
upregulate tumour associated antigens (Ags) in the context of major histocompatibility antigen (MHC)
class I.2;5 The generation of tumour specific CTLs primed by tumour Ag loaded dendritic cells (DCs) is
a promising strategy that is currently being investigated. This treatment is based on the abilities of DCs
to process and present Ags to naive T cells, in addition to expressing co-stimulatory signals that drive
their differentiation and expansion.5-12 This paper will assess the opportunities and major challenges
presented by Phase I clinical studies, to determine whether DC based immunotherapy can be
successfully used to mount an effective immune response against tumours in colorectal cancer.
Dendritic Cells (DCs)
DCs, the most potent Ag presenting cells in vivo, arise from CD34+ progenitors in the bone
marrow and blood, and their expansion and differentiation is influenced by cytokine growth factors.6-9
[3]
The progenitors seed lymphoid tissues and develop into immature DCs with enhanced Ag uptake and
processing capacities (Figure 1).6-8 Upon stimulation by inflammatory cytokines, they become mature
DCs optimized for migrating to lymph nodes and priming naive T cells in an MHC (i.e., human
leukocyte antigen, HLA) restricted manner (Figure 1).6-8 The maturation state of DCs modulates their
capacity for immune stimulation,6-8 and murine studies that have had success in cancer immunotherapy
primarily used mature DCs.7;10;11 Immature DCs have been shown to limit the immune response by
inducing the generation of CD4+CD25+ “regulatory” T cells.6;12 Of the studies reviewed here,
immature DCs were only used by one group (Table 1), who also co-injected the cytokines TNF and
IFN to induce DC maturation in vivo.13 As DCs derived from colorectal cancer tissues are defective in
Ag presenting and T cell stimulating functions, and chemotherapy/radiation treatments are
immunosuppressive,9;14 autologous patient DCs were expanded and loaded with Ag ex vivo.
[4]
Figure 1: Phenotypic and functional differences between immature and mature dendritic cells
(DCs)
The activation state of DCs can be modulated by inflammatory stimuli (lipopolysaccharide, LPS),
cytokines (tumor necrosis factor TNF-; IL-10), or growth factors (vascular endothelial growth
factor, VEGF).
Immature DCs are primed for antigen uptake and processing, whereas mature DCs have enhanced
expression of antigen presenting, adhesive, and T cell co-stimulatory molecules.
Adapted from: Timmerman JM, Levy R. Dendritic cell vaccines for cancer immunotherapy.
Annu.Rev.Med. 1999;50:507-529.
[5]
DC based immunotherapy
In preclinical studies, Ag-loaded DCs elicited protective tumour immunity and tumour-specific
CTL responses against both primary and metastasized tumours.9;11;15 Most of these studies introduced
genes encoding foreign, “model” Ags into mouse tumours, which greatly enhanced tumour
immunogenicity.9;11;15 In humans, tumour specific Ags have been identified and used with some
success in hematologic malignancies and melanoma.2;5-7;10 These Ags include gene products that are
silent in normal tissues but overexpressed in tumours or reactivated embryonic gene products.2;10
Several colorectal tumour associated Ags (TAAs) have been described,2;6;7 including Mage-3 and
human carcinoembryonic Ag (CEA), self-derived, weakly immunogenic TAAs that are also detected,
at much lower levels, in non-cancerous tissues.7;8;10 The efficient endocytotic and T cell co-stimulatory
properties of DCs presents a useful means of overcoming tolerance to these TAAs.
DC based immunotherapy was successfully demonstrated in a murine colorectal cancer model
using transgenic CEA-expressing mice.15;16 Specifically, subcutaneous injections of DCs adenovirally
transduced with CEA induced significant cytotoxic activity of spleen cells against CEA-expressing
tumours, and resulted in eradication of subcutaneous tumours in 80% of mice.16 Based on the
preclinical success, Phase I studies have been conducted in small numbers of late stage colorectal
cancer patients (mean n: 10, Table 1 – attached as supplementary file) to determine the safety and
feasibility of DC based immunotherapy.13;17-25 In vitro assays of immune function allowed evaluation
of pre- and post-vaccination tumour immunity in these patients. Radiologic evaluations of tumour
burden, quantification of serum TAA levels, and rates of disease progression were used as clinical
markers of response to treatment. The following sections will discuss the successes and limitations
highlighted in the Phase I studies, in light of data obtained on: a) feasibility; b) tolerability; c) induction
of tumour specific immune response; and d) positive clinical outcomes for this treatment.
[6]
DCs isolated from colorectal cancer patients can be expanded and activated ex vivo
As DCs comprise less than 1% of leukocytes in blood,6-8;11 obtaining sufficient amounts for
vaccination presents an obstacle for human studies, although significant advances to overcome this
issue have been achieved. The common method consisted of leukapheresis to isolate adherent
peripheral blood mononuclear cells (PBMC), followed by in vitro culture in the presence of cytokines,
i.e., granulocyte-monocyte colony-stimulating factor (GM-CSF) and IL-4, to induce differentiation of
CD14+ monocytes into DCs (Figure 2).6-8;12 Alternatively, CD34+ hematopoietic progenitor cells,
which maintain their proliferative ability and can be expanded in larger numbers, were purified from
leukapharesis products, and cultured with a cocktail of cytokines, including IL-6, IL-3, and the growth
factor Flt-3 ligand, followed by differentiation with GM-CSF and IL-4.6-8;12 These protocols have
allowed the isolation of up to 500 million DCs from patients, with approximately 5 million DCs used
per vaccination.12 To further enhance DC yield and stimulate their maturation, injections of Flt-3 ligand
before PBMC collection have been successfully used in animal studies,12;26 and applied in one19 of the
Phase I studies.
[7]
Figure 2: Basic protocol for DC culture for use in cancer immunotherapy
+ Maturation signal (TNF
Vaccine production involves collection of blood from cancer patients (Day 0). Typically, leukapheresis is used
to isolate large numbers of peripheral blood mononuclear cells (PBMC). Autologous DCs are harvested from
CD14+ monocytes by culturing PBMC in the presence of cytokines (interleukin 4, IL-4; granulocyte
macrophage-colony stimulating factor, GM-CSF) for ~7 days. This is followed by ex vivo exposure of the DCs
to maturation signals (tumor necrosis factor , TNF-) and tumour antigen (Day 8), for return of pulsed
DCs to the patient (Day 9).
Adapted from: Chen W, Rains N, Young D, Stubbs RS. Dendritic cell-based cancer immunotherapy:
potential for treatment of colorectal cancer? J.Gastroenterol.Hepatol. 2000;15:698-705.
[8]
Cultured DCs can be loaded with antigenic variants of peptides expressed by colorectal tumours
To overcome tolerance to the weakly immunogenic colorectal cancer TAAs, cultured DCs were
primed with antigenic fragments of CEA and Mage-3 ex vivo. Earlier phase I studies identified the
recombinant and healthy control derived CEA peptide fragments CAP-1 and CEA652, which generated
HLA-A2.1 and HLA-A24 restricted CTLs, respectively, in vitro.13 To improve TAA-HLA affinity and
facilitate HLA-T cell receptor interactions,12 CAP-610D, a CEA derivative with an altered peptide
sequence, was also used in two of the current studies.22;24 As these strategies require knowledge of the
Ag peptide sequence and restrict the use of patients with corresponding HLA alleles,12 clinical trials of
other malignancies have also used DCs loaded with tumour cell lysates or their messenger RNA
content, or malignant cells fused with DCs.5-12 To enhance their Ag presenting capacity, DCs were also
packaged with adjuvants (i.e., Keyhole Limpet Hemocyanin, KLH) to stimulate helper CD4+ T
cells,11;19;24;25 cytokine genes (i.e., TNF, IFN),13 or co-stimulatory molecules (i.e., synthetic CpG
oligodeoxynucleotides)23(Table 1). Although a direct comparison of the immunogenicity of the
different Ags has not been done, the presence of adjuvants or co-stimulation was previously shown to
enhance tumour specific CTL responses compared to Ag loaded DCs alone.2;5;6;10
DCs harvested from colorectal cancer patients have normal morphology and phenotype
Following culture, DCs were verified to be sterile, >50% pure by flow cytometric analysis of
HLA-DR and CD86 expression, and >70% viable by trypan blue exclusion.13;17-25 Cultured cells had
the characteristic stellate morphology (Figure 3), and their phenotype was tested by quantifying
expression levels of DC markers by flow cytometry (Figure 4).13;17-25 There was a high level of
heterogeneity in the phenotype of DCs isolated from different patients even within studies, 13;17-25 which
[9]
may reflect differences in immune competence of patients as a result of radiation or chemotherapy.
Although their use has not been standardized in clinical trials, comparisons of DCs from different
sources suggested that CD34+ cell derived DCs are more potent in their ability to activate T cells.27
Figure 3: Cultured DCs generated from colorectal cancer patients have normal morphology
Adherent peripheral blood mononuclear cells were obtained from a patient with advanced colorectal cancer and
harvested for DCs. Diff-Quick staining of the cultured DCs (arrows) show many characteristic widespread
cytoplasmic projections.
Adapted from: Chen W, Rains N, Young D, Stubbs RS. Dendritic cell-based cancer immunotherapy:
potential for treatment of colorectal cancer? J.Gastroenterol.Hepatol. 2000;15:698-705.
[10]
Figure 4: Cultured DCs generated from colorectal cancer patients have normal phenotype
Surface marker expression of cultured DC
preparations from a representative patient was
analyzed by flow cytometry.
DCs obtained on day 6 (dark areas) and after
incubation with tumor necrosis factor- (TNF-)
on day 8 (white areas) were stained with
monoclonal antibodies against HLA-DR, CD86,
CD80, CD83, CD14, CD40, and isotype-matched
monoclonal antibody controls.
Marker Day 6 DCs Day 8 DCs
96 ± 4.9
95.7 ± 5.3
DR
98 ± 1.7
98.2 ± 3.0
CD86
26.8 ± 11.6 57.7 ± 19.6
CD80
16 ± 7.5
58.9 ± 17.0
CD83
99.4 ± 0.4
97.2 ± 4.3
CD40
20.2 ± 16.0 7.8 ± 9.9
CD14
Dendritic cell surface marker expression
(mean ± SD)
Quantification of surface expression
markers showed that Day 6 DCs had
a typical immature DC staining
profile: HLA-DR+, CD86+, CD80low,
CD14low, and CD83low. Day 8 DCs
generally displayed a more mature
DC phenotype: HLA-DR+, CD86+,
CD80+, CD14–, CD40+, and CD83+.
Adapted from: Liu KJ, Wang CC, Chen LT et al. Generation of carcinoembryonic antigen (CEA)specific T-cell responses in HLA-A*0201 and HLA-A*2402 late-stage colorectal cancer patients after
vaccination with dendritic cells loaded with CEA peptides. Clin.Cancer Res. 2004;10:2645-2651
[11]
The route and dosage of DC vaccinations requires optimization
To date, the route of DC administration that allows them to efficiently home to secondary
lymphoid organs and activate T cells has not been addressed. In the current studies, DCs were injected
intravenously (IV), intradermally (ID), subcutaneously (SC), and intranodally (IN) (Table 1).13;17-25 It
was not possible to determine which injection mode led to optimal immune response generation, based
on the level of heterogeneity between the small number of studies. Previous comparisons of IV, ID, and
SC DC administrations suggested that ID injections may improve CTL generation.28;29 However, in
murine models, less than 5% of ID injected DCs were shown to actually migrate to draining lymph
nodes, although preconditioning the injection site with inflammatory cytokines enhanced DC
trafficking.12;29;30 Mouse models also showed promising results for intra-tumoural (IT) injections of
DCs pre-transfected with immunostimulatory cytokines (i.e., IL-12), which were able to eradicate
transplanted colorectal tumours.31 In humans,
111
Indium labelling of cytokine transfected DCs showed
that, following IT injections, all the DCs remained inside the lesions, regardless of their maturation
status.28 Tumour cells actively retained DCs by secreting IL-8, which bound to the DC receptors
CXCR1 and CXCR2.28 Further studies are needed to directly compare the effectiveness of different
injection routes, and to delineate the cytokines/adhesive factors that promote recruitment to tumour
sites and migration to lymphoid tissues.
The minimum DC dose needed to generate enough effector CTLs is also unknown, and varied
greatly between the current studies (Table 1). Fong et al administered increasing DC doses to three
cohorts of patients, but a possible correlation with immune or clinical response was not explored. 19
Another level of heterogeneity was introduced by differences in the frequency and total number of
immunizations (Table 1). This remains an important question to address, as previous studies reported
that frequent T cell stimulation can lead to activation induced T cell death.9
[12]
DC based immunotherapy is well tolerated by colorectal cancer patients
As treatment associated toxicity is a major limitation of current therapies, it was important to
verify that injections of TAA loaded DCs did not cause adverse effects, including induction of T cell
mediated autoimmunity through the presentation of self Ags. In a murine model of colorectal cancer,
no autoimmunity was detected for up to one year after treatment by a CEA vaccination regimen.32 The
current studies also ruled out the generation of autoimmune responses. There was also no evidence of
hepatic, renal, or pulmonary toxicity assessed by World Health Organization (WHO) Criteria or the
National Cancer Institute (NCI) common toxicity scale.13;17-25 The most common side effects were
transfusion-like reactions that manifested as self resolving diarrhoea and fever (NCI grade I/II in
severity) (Table1). All other symptoms were described in single cases and were postulated to be due to
advanced disease.
Injected DCs can stimulate TAA specific immune response
The ability of DC vaccines to stimulate a TAA specific immune response is the commonly used
biological marker of treatment effectiveness. Although other immune cells like monocytes and natural
killer cells may also have roles in tumour immunity, the current studies focused mainly on in vitro
quantitative and functional assays of newly generated CTLs. Preliminary screening for immune
response generation was typically done by delayed type hypersensitivity (DTH) skin tests.13;17;18;20;22;25
The diameter of dermal induration/erythema after injection of the TAA alone or Ag loaded DCs was
measured after 48-72 hours. Skin indurations greater than 5 mm in diameter were typically defined as
positive responses, and control Ag loaded DCs or DCs alone were used to verify TAA specificity of the
DTH reactions. By these criteria, a positive DTH response was evident in a majority of the patients
[13]
tested, and immunostaining of injection site biopsies confirmed T cell infiltration (Figure 5, Table 1). It
is unclear whether all responses were truly Ag specific, as vaccinations of DCs alone caused
indurations in some patients, and indurations in the absence of TAA specific CTLS were detected in
others.25 Conversely, staining of skin biopsies showed T cell infiltration in the absence of positive DTH
tests.18 Interestingly, one group observed local erythemas in 80% of patients on initial vaccination with
CEA derived peptide loaded DCs,25 which implies that some form of anti-CEA immunity was already
present in these patients.
Figure 5. Delayed Type Hypersensitivity (DTH) reaction at the DC vaccination site showed
evidence of immune response generation
The photograph (a) shows the vaccinated inguinal site of a representative patient 48 hours post
injection. Erythema and induration ~30 mm in diameter were observed at the vaccinated site.
Histological analysis of the skin biopsy specimen (b) showed massive infiltration of lymphocytes at
the vaccinated site.
Adapted from: Matsuda K, Tsunoda T, Tanaka H et al. Enhancement of cytotoxic T-lymphocyte
responses in patients with gastrointestinal malignancies following vaccination with CEA peptidepulsed dendritic cells. Cancer Immunol.Immunother. 2004;53:609-616.
[14]
The ability of newly generated CTLs to lyse TAA expressing cells was tested in cytotoxicity
assays.13;17;19;20;24 Typically, patient PBMCs were stimulated with autologous TAA-loaded DCs to
generate CTLs, which were then quantified by their ability to lyse
51
Cr labelled target cells. After
vaccination, >50% of patients tested showed CTL mediated cell lysis of TAA expressing tumour cell
lines, or other cell lines pulsed with the TAA of interest (Figure 6, Table 1). One study confirmed that
cell lysis was CTL mediated and MHC restricted by showing inhibition of cytotoxicity by antibodies
against MHC class I and CD8, but not by antibodies against MHC class II and CD4.17 Despite the
widely reported positive response, patient PBMCs had to undergo multiple rounds of TAA
stimulation13;17;19;20;24 to generate sufficient amounts of Ag specific CTLs for testing.
[15]
Figure 6: DC vaccinations generated tumour associated antigen (TAA) specific cytotoxic effector
T cells
DC stimulated CTLs against TAA were obtained from a representative patient’s PBMC preparation,
before (top) and after vaccination (bottom).
The cytotoxicity of CTLs were assayed against the tumor cell lines COLO 205 (colorectal cancer cell
line) and control, K-562 (chronic myelogeous leukemia cell line). Testing was done at various effector
cell (E) to target cell (T) ratios (3:1, 10:1, 30:1).
Post vaccination, the patient’s CTLs showed enhanced lysis of the TAA expressing cell line.
Adapted from: Babatz J, Rollig C, Lobel B et al. Induction of cellular immune responses against
carcinoembryonic antigen in patients with metastatic tumors after vaccination with altered peptide
ligand-loaded dendritic cells. Cancer Immunol.Immunother. 2006;55:268-276.
[16]
Analyses of TAA specific CTL activation were typically confirmed by monitoring their
cytokine production by ELISPOT and/or intracellular cytokine staining.17;18;20-25 Dilutions of TAA
loaded autologous DC were used to activate CTLs from patient PBMC to stimulate cytokine secretion.
TAA specific CTL increases were seen in ~50% of patients evaluated by these assays (Figure 7, Table
1). The only study with negative results used Mage-3 derived peptide loaded DCs in the absence of any
co-stimulatory molecules.20 Like in the cytotoxicity assays, multiple re-stimulation of PBMC were
required in all but one study that used DCs genetically modified with a recombinant poxvirus vector
encoding CEA and co-stimulatory molecules.22 Two groups also showed the generation of TAA
specific helper T cells based on cytokine secretion and expression of the surface marker CD4.20;22
[17]
Figure 7: DC vaccinations generated TAA specific IFN producing T cells
DC stimulated PBMCs were sampled from a representative patient, during 2 cycles of DC
immunization.
The numbers of activated, IFNproducing T cells generated in response to either the target TAA
(triangles) or irrelevant, control Ag (circles) in each patient sample, are shown.
In this patient, frequencies of TAA specific T cells increased during the first cycle of immunizations,
and stayed relatively stable during the second cycle of therapy.
Adapted from: Morse MA, Clay TM, Hobeika AC et al. Phase I study of immunization with dendritic
cells modified with fowlpox encoding carcinoembryonic antigen and costimulatory molecules.
Clin.Cancer Res. 2005;11:3017-3024.
[18]
The MHC tetramer assay was also used by some studies to measure frequencies of CD8+ T
cells. This assay did not require re-stimulation to yield detectable Ag specific CTLs, although
fluorophore-tagged MHC tetramers (matched for each patient’s HLA allele) had to be pre-loaded with
the relevant Ag for exposure to patient PBMC.17-19;23;25 By this method, the percentage of peripheral
blood CTLs that reacted specifically with the TAA-MHC tetramers increased from baseline values up
to 1% (Figure 8, Table 1). This level of Ag specific CTLs was previously shown to be sufficient to
cause tumour remission in murine tumour models.33 At present, it is difficult to compare these results
(expressed as “% of CD8+ T cells that are TAA specific”) with those obtained in ELISPOT assays (“%
of total PBMC that are TAA specific”),9 which calls for some standardization in reporting results for
comprehensive analysis.
Taken together, moderate to strong CTL responses against target TAAs were demonstrated in
all studies, although low amounts of CTLs in patient PBMC necessitated multiple rounds of restimulation. The timing and rounds of stimulation varied significantly between studies. The degree to
which these expanded T cell populations reflect T cell immunity in vivo is unclear. Interestingly,
Lesterhuis et al showed that, compared to peripheral blood, abdominal lymph nodes, and resected
tumour tissue, post treatment DTH skin biopsies may yield larger number of TAA specific CTLs for
functional/phenotypic analyses, without the need for re-stimulation.25 Another limiting factor in the
studies was the use of peripheral blood samples to assay CTL phenotype/function, as these samples
might not accurately represent what actually happens in the tumour milieu. Additional studies are
needed to evaluate post DC vaccination immune responses in secondary lymphoid organs, or in the
tumour itself.
[19]
Figure 8: DC vaccinations resulted in increased frequencies of TAA specific T cells
CD8+ cells
Fluorophore-tagged MHC tetramers (matched for patient HLA allele) were loaded with the tumour
associated antigen (Carcinoembryonic Antigen derived peptide: CAP-1) or control antigen (Human Tcell lymphotrophic virus type I: HTLV-1) for exposure to patient PBMC. Data were obtained before
(baseline) and after (post therapy) DC treatments.
The representative patient showed some CAP-1 specific response at baseline (0.045% CD8+ T cells),
which increased (0.190% CD8+ T cells) following therapy.
Adapted from: Weihrauch MR, Ansen S, Jurkiewicz E et al. Phase I/II combined chemoimmunotherapy
with carcinoembryonic antigen-derived HLA-A2-restricted CAP-1 peptide and irinotecan, 5fluorouracil, and leucovorin in patients with primary metastatic colorectal cancer. Clin.Cancer Res.
2005;11:5993-6001.
[20]
TAA loaded DCs did not consistently induce clinically relevant outcomes
The ability to generate clinically relevant tumour responses is the critical determinant of
treatment effectiveness. In the current trials, patients were monitored by imaging studies (computed
tomography (CT) scans, and chest radiographs) to measure tumour burden.13;17-25 Decreases in serum
levels of the TAAs (CEA/MAGE-3) were monitored as surrogate markers of treatment response.13;17-25
Of the 100 patients enrolled in the studies, the disappearance of all tumour, or “complete response” to
treatment, was reported in 12; however, 6 of the responders were from one study that also used
chemotherapy as part of the treatment regimen (Table 1).13;17-25 “Partial response,” which had different
definitions and represented some degree of tumour regression/metastasis resolution (Figure 9), was
seen in 3 patients, whereas stable disease was noted in 21 patients (Table 1).13;17-25 These numbers
should be interpreted with caution; there were large variations in the assessment of clinical responses,
and it was not explicitly stated whether data collection and/or analysis was blinded. In addition, follow
up times in these studies were fairly short, with an average duration of 3 months.13;17-25 Decreased TAA
levels were only detected in 5 of the patients evaluated and did not significantly correlate with tumour
regression (Table 1). As a majority of treated patients went on to have progressive disease, the
treatment appears to have, at best, modest clinical benefits in these Phase I studies.
Overall, there was no statistically significant correlation between immunologic and clinical
outcomes, which may be related to the small numbers of patients used in these studies. Alternatively,
none of the currently available assays may actually measure function with direct relevance to how
tumours are attacked immunologically in the body. At this point, it is difficult to ascertain whether the
observed responses were elicited by a TAA specific immune response, or by an immune-stimulatory
effect of the adjuvants alone. For future studies, data on time to disease progression and annual/overall
[21]
survival rates as a function of immunotherapy, and the use of standardized criteria to judge clinical
outcomes, should provide useful information.
Figure 9. DC treatments led to some improvements in clinical symptoms
Computed tomography scans were performed on a representative patient 1 month before the first
vaccination (Before treatment) and 4 months after the final vaccination (After treatment). The patient
did not receive any other concurrent treatments.
Following DC vaccination, this patient developed resolution of growing lung metastases (red arrow)
and malignant pleural effusion (white arrow).
Adapted from: Fong L, Hou Y, Rivas A et al. Altered peptide ligand vaccination with Flt3 ligand
expanded dendritic cells for tumor immunotherapy. Proc.Natl.Acad.Sci.U.S.A 2001;98:8809-8814.
[22]
Obstacles to clinical translation
These pilot studies showed that DC immunization was well tolerated and induced Ag specific
immune responses in late stage colorectal cancer patients. However, the clinical response rate was
moderate and variable. The ability to culture and mature large numbers of functional DCs, and the
identification of immunogenic TAA derivatives for priming DCs represent major achievements.
However, there appear to be a number of areas that need optimization, including formulation of the
vaccine (i.e., use of adjuvants/cytokines/co-stimulatory molecules), populations of DCs used to allow
optimal co-stimulation of T cells, type of Ag used, and the route, dose, and frequency of DC
administration. Indeed, given the large number of issues that still need to be ironed out, it is surprising
that any clinical responders were observed at all. As discussed above, there were differences in
sensitivities and interpretations of the immunologic assays used to compare T cell frequencies.9;33 The
high level of heterogeneity across studies thus indicates a need to establish standardized immune assays
to permit meta-analysis. Additionally, the current studies used short term vaccinations carried out over
a few months; longer follow up times with revaccination strategies should provide more information.
Tumour modulation of the immune response presents a major obstacle to DC based
immunotherapy, especially in light of the large tumour burdens in the patients used in these studies.
Tumour cells can actively suppress the immune system through the production of immunosuppressive
cytokines (i.e., TGF-
-10), which inhibit DC maturation and function.5-7;15 Although culture and
expansion of DCs ex vivo avoided the maturation hurdles, injected DCs may have been rendered
“dysfunctional” once they reached the immunosuppressive tumour environment. Secondly, even if
TAA specific CTLs were generated, the genetic instability of cancer cells may have allowed them to
acquire mutations to downregulate their HLA and/or TAAs expression, allowing them to escape
immune surveillance.5-7;15 Based on the inherent diversity in tumour behaviour, a standard vaccination
[23]
protocol may not be possible at all, and patients may require customized quantities and potencies of
infused DCs.
The relative lack of information from the current studies makes it ethically impossible to design
larger trials that include patients with early stage colorectal cancer. However, these studies are limited
in their generalizability; as the patients had increased tumour burden and chemotherapy induced
immune-suppression, their immune response post DC vaccination may have been overwhelmed by
immunosuppressive factors secreted by their tumours. A recent systematic review of immunotherapy
for colorectal cancer showed that subgroups of patients may benefit from this treatment after tumour
resection.34 DC based immunotherapy may similarly prove more valuable for recently diagnosed
patients or those who have recently undergone surgery/chemotherapy to reduce their tumour burden.
Conclusion
The minimal adverse response to treatment, success in preclinical studies, and induction of
measurable TAA specific T cells post immunization, makes DC based immunotherapy potentially
promising. Although a host of unanswered questions remain about tumour-immune system interactions,
and additional issues are still being unearthed, data from the Phase I trials have provided important
insights that can be used to design larger, standardized trials. A high degree of optimization of
vaccination regimens and outcome assessments is needed in future studies. This should allow
systematic review of results to ultimately reach a consensus on the clinical efficacy of DC based
immunotherapy in colorectal cancer, which will have important implications for the millions of
individuals suffering from this disease.
[24]
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