Treatment of patients with locally advanced cancer
still poses a major problem for clinicians. Patients with locally advanced
tumors have shown a poor prognosis when treated by surgery and/or radiotherapy
alone. Using these two treatment modalities local control can be reached in a
number of patients, but distant metastases usually appear shortly after removal
of the primary tumor. This observation was made after local treatment of several
tumor types, including breast cancer, esophageal cancer, stomach cancer, bladder
cancer, and head and neck cancer, thus implying that distant micrometastases are
already present at the time of diagnosis of a locally advanced tumor in most
patients. The addition of systemic adjuvant and later also neoadjuvant
chemotherapy showed an improvement of prognosis for these patients. Adjuvant
chemotherapy improves survival in patients with colon and breast cancer.
Adjuvant active specific immunotherapy with an autologous tumor cell-bacille
Calmette-Guérin (BCG) vaccine improved disease-free survival in patients with
stage II colon cancer.
Neoadjuvant or primary chemotherapy improves organ preservation in patients
with head and neck carcinomas without compromising survival. This approach,
often combined with radiotherapy, has also been tested in bladder,[5,6]
esophageal,[7,8] and gastric cancer. Although down-staging is often possible
with this approach, no survival benefit has been demonstrated so far. We tried
to make use of the advantages of neoadjuvant chemotherapy by combining it with
immunotherapy in the treatment of locally advanced cancer. Because the tumor
antigens that are capable of inducing a specific immune response are unknown for
many tumor types, we chose to test an autovaccination approach in the form of
prolonged neoadjuvant chemotherapy combined with immunomodulating cytokines. In
order to study immunologic and biologic events during treatment of locally
advanced tumors by a new multidisciplinary approach, we selected locally
advanced breast cancer as a model. Patients with locally advanced breast cancer
have a primary tumor and regional lymph nodes, which are relatively easily
accessible for sequential biopsies in order to study tumor biology.
Locally advanced breast cancer includes patients with stage IIB (> 5 cm in
diameter), IIIA, and IIIB tumors, according to the American Joint Committee on
Cancer (AJCC) guidelines. This results in a heterogeneous group of breast
neoplasms with different biologic and clinical characteristics. Patients
present with large primary tumors and no signs of distant metastases at the time
of diagnosis. Increasing tumor size may result in increased heterogeneity and
resistance to treatment. When treated with local therapy alone, patients
with locally advanced breast cancer have a poor prognosis; the 5-year overall
survival rate is 20%.[13-15] For patients with inflammatory breast cancer the
5-year overall survival rate is even worseonly 5%.[16-18] When treated with
local therapy alone, systemic metastases tend to develop rapidly, indicating the
existence of micrometastases in most patients at diagnosis. By means of
immunohistochemistry or reverse polymerase chain reaction assay, cancer cells
can be detected in blood and bone marrow in up to 30% of patients with
early-stage breast cancer at time of surgery.[19,20]
Systemic treatment with chemotherapy alone fails to achieve local control,
due to either primary drug resistance or induced drug resistance of the
tumor cells. Combined-modality treatment with neoadjuvant chemotherapy has
significantly improved the survival rate of patients with locally advanced
breast cancer over the past 2 decades. The aim of this approach is to render
patients with large tumors operable and to eradicate micrometastases at an early
stage, when they are still sensitive to chemotherapy.[10,23,24] Present
treatment schedules usually consist of three to four conventional-dose
neoadjuvant chemotherapy cycles prior to local therapy by means of surgery
and/or radiotherapy, often followed by a number of adjuvant postoperative
chemotherapy cycles.[10,23] Clinical response rates range between 30% and 80%,
with 10% to 30% clinical complete remissions. The 4- or 5-year disease-free
survival rates vary from 33% to 40%, and the 5-year overall survival rate varies
from 40% to 60% with long-term survival in 15% of patients.[25-28]
Doxorubicin and cyclophosphamide (Cytoxan, Neosar) are among the most
effective chemotherapeutic agents in breast cancer, with reported response rates
of 25% to 50% when used as single agents and 45% to 60% when in standard
combinations. For both agents, investigators have observed immunomodulating
properties enhancing antitumor immunity. Because of increasing evidence of a
steep dose-response effect in breast cancer for a number of chemotherapeutic
agents, including doxorubicin and cyclophosphamide,[30-34] we developed a
dose-intensive regimen for doxorubicin plus cyclophosphamide.[32,35]
Earlier, we reported the results of a study of 42 patients with locally
advanced breast cancer treated with four to six cycles of neoadjuvant
chemotherapy with moderately high-dose cyclophosphamide, doxorubicin, and
granulocyte-macrophage colony-stimulating factor (GM-CSF [Leukine]) prior to
surgery and postoperative radiotherapy. GM-CSF was chosen as a hematopoetic
growth factor instead of the less toxic granulocyte colony-stimulating factor
(G-CSF [Neupogen]) because of the former’s reported immunostimulatory
effects.[23,35,36] This regimen showed a high clinical response rate of 98%,
with 50% clinical complete remissions. Remarkably, analysis of the data showed a
trend toward improvement in disease-free and overall survival in patients who
received more chemotherapy cycles. The 3-year disease-free survival rates are
0%, 54%, and 67%, respectively, for patients who received four, five, and six
cycles. The 3-year overall survival rates are 20%, 62%, and 79%, respectively,
for patients who received four, five, and six cycles (see Figure
We hypothesized that these promising results are not only related to the 50%
increase in the chemotherapy dose, but also related to biologic and immunologic
consequences of the prolonged presence of the primary tumor and draining lymph
nodes in situ. Release of antiangiogenic factors was shown in a preclinical
model of the Lewis lung carcinoma. Such factors were shown to inhibit the growth
of distant metastases. Circulating angiogenesis inhibitors produced by the
primary tumor in locally advanced breast cancer patients, for instance, might
inhibit the growth of micrometastases and add to the therapeutic effects of
prolonged neoadjuvant chemotherapy. The long-term administration of GM-CSF might
have resulted in the stimulation of antigen-presenting cells and cytotoxic T
cells, thus enhancing the antitumor immune response.[23,36]
For many years investigators have been focusing mainly on the direct
antitumor and toxic effects of different chemotherapeutic agents, while biologic
and immunologic factors inherent to the primary tumor might also add to the
therapeutic effects. In order to study these events we have initiated the
Spinoza trial, an international randomized clinical trial of locally advanced
breast cancer patients comparing six neoadjuvant cycles to three neoadjuvant
cycles combined with three adjuvant cycles, and GM-CSF to G-CSF administration
(see Figure 2). This study investigates immunologic and biologic parameters in
biopsy and mastectomy specimens and in the peripheral blood of all patients. The
biologic and immunologic consequences of the prolonged presence of the primary
tumor and its draining lymph nodes, prolonged neoadjuvant chemotherapy, and
prolonged GM-CSF administration will be described in further detail below.
In recent years more attention has been paid to the function of the immune
system in cancer patients, and the possibility of enhancing the antitumor
effects of the immune system. A number of tumor-associated antigens
overexpressed in breast tumors have been identified. These include HER2/neu, CEA,
and MUC1. These antigens have been shown to induce T-cell responses in healthy
donors and breast cancer patients.[37-40] T-cell responses are induced by
presentation of tumor antigens by professional antigen-presenting cells. These
include dendritic cells, macrophages/monocytes, and B cells. Dendritic cells
are widely regarded as the most professional and potent antigen-presenting
cells. They play a key role in the initiation of an antitumor immune
response.[41-44] Dendritic cells are derived from bone marrow progenitor cells.
They produce circulating precursors that migrate to the tissues, where they
remain as immature cells with high phagocytic capacity. After phagocytosis and
processing of antigens these cells migrate to the lymphoid tissues. They present
antigens on their cell surface in association with MHC-I and MHC-II molecules.
Dendritic cells are able to activate CD8-positive cytotoxic T lymphocytes and
CD4-positive T cells. Dendritic cell infiltration in the primary tumor has been
associated with a reduced incidence of metastatic disease and prolonged
survival, for several tumor types.[45-50]
In patients with breast cancer a pattern of compartmentalization of immature
and mature dendritic cells characteristic of active immune response has been
observed. Immature dendritic cells are found in the centers of tumors, while
mature dendritic cells are found in the peritumoral areas and lymphoid tissues
surrounded by clusters of T cells. Tumor-specific T cells have been found in
cancer patients, but they fail to control tumor growth. This can be explained by
the existence of mechanisms for evading immune surveillance. A number of studies
have suggested the existence of immunosuppressive conditions in patients with
various tumors. Several cytokines produced by tumor cells, such as
interleukin (IL)-10, transforming growth factor (TGF)-beta, IL-6, and vascular
endothelial growth factor (VEGF), have been shown to inhibit dendritic cells’
maturation in vitro. IL-10 and TGF-beta have also shown inhibition of T-cell
effector functions in vitro.[51-55] Tumor-induced Fas-mediated early apoptosis
of dendritic cells and T cells has also been reported.
In patients with locally advanced breast cancer, investigators have found a
dysfunction of dendritic cells and T lymphocytes. Dysfunction of T lymphocytes
in breast cancer patients was found to be caused by a functional disturbance of
dendritic cells. It was shown that T cells from breast cancer patients can be
stimulated by dendritic cells of healthy controls.[41,57] In more advanced
stages of breast cancer, depression of T-cell reactivity becomes progressive and
is significantly related to survival. Dendritic cells are optimally equipped
to activate T cells. Prolonged administration of GM-CSF in combination with
neoadjuvant chemotherapy might help dendritic cells to overcome these
immunosuppressive conditions present in breast cancer patients.
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